MCP-1 binding nucleic acids

ABSTRACT

The present invention is related to a nucleic acid, preferably binding to MCP-1, selected from the group consisting of Type 1A nucleic acids, type 1B nucleic acids, Type 2 nucleic acids, Type 3 nucleic acids, Type 4 nucleic acids and nucleic acids comprising SEQ ID NOs:87-115

The present invention is related to nucleic acids binding to MCP-1, andthe use thereof for the manufacture of a medicament and a diagnosticagent, respectively.

Human MCP-1 (monocyte chemoattractant protein-1; alternative names, MCAF[monocyte chemoattracting and activating factor]; CCL2; SMC-CF [smoothmuscle cell-colony simulating factor]; HC-11; LDCF; GDCF; TSG-8; SCYA2;A2; SwissProt accession code, P13500) was characterized by three groupsindependently (Matsushima 1988; Rollins 1989; Yoshimura 1989). Itconsists of 76 amino acids and features a heparin binding site like allchemokines. The two intramolecular disulfide bonds confer a stable,rigid structure to the molecule. Furthermore, MCP-1 carries apyroglutamate at its amino terminus. At Thr 71, a potential O-linkedglycosylation site is located. Additional MCP family members exist bothin humans (MCP-2, -3, -4) and mice (MCP-2, -3, -5). The human proteinsare approximately 70% homologous to human MCP-1.

The structure of MCP-1 has been solved by NMR (Handel 1996) and X-ray(Lubkowski 1997). The MCP-1 monomer has the typical chemokine fold inwhich the amino-terminal cysteines are followed by a long loop thatleads into three antiparallel β-pleated sheets in a Greek key motif. Theprotein terminates in an a helix that overlies the three β sheets (PDBdata accession code 1DOK).

Although the three-dimensional structure of MCP-1 forms from differentmammalian species has generally been maintained, the amino acid sequencehas not particularly well been conserved during evolution. Sequencealignment results demonstrate 55% overall sequence similarity betweenhuman and murine MCP-1 (also called JE) within the first 76 amino acids.Apart from the amino acid sequence, murine MCP-1 differs from humanMCP-1 in molecular size (125 amino acids) and the extent ofglycosylation. Murine MCP-1 contains a 49-amino acid carboxyterminaldomain that is not present in human MCP-1 and is not required for invitro bioactivity. Human MCP-1 shares the following percentage ofidentical amino acids with MCP-1 from:

-   -   Macaca mulatta (Rhesus monkey) MCP-1 97%    -   Sus scrofa (Pig) MCP-1 79%    -   Equus caballus (Horse) 78%    -   Canis familiaris (Dog) MCP-1 76%    -   Oryctolagus cuniculus (Rabbit) MCP-1 75%    -   Bos Taurus (Bovine) 72%    -   Homo sapiens MCP-3 71%    -   Homo sapiens Eotaxin 64%    -   Homo sapiens MCP-2 62%    -   Mus musculus (Mouse) MCP-1 55%    -   Rattus norvegicus (Rat) MCP-1 55%

Given this high degree of divergence it may be necessary to generateantagonists of rodent MCP-1 for successful performance ofpharmacological studies in rodent models.

MCP-1 is a potent attractor of monocytes/macrophages, basophils,activated T cells, and NK cells. A wide variety of cell types, such asendothelial cells, epithelial cells, fibroblasts, keratinocytes,synovial cells, mesangial cells, osteoblasts, smooth muscle cells, aswell as a multitude of tumor cells express MCP-1 (Baggiolini 1994). Itsexpression is stimulated by several types of proinflammatory agents suchas IL-1β, TNF-α, IFN-γ, LPS (lipopolysaccharide), and GM-CSF.

Rather unusual in the promiscuous chemokine network, MCP-1 is highlyspecific in its receptor usage, binding only to the chemokine receptorCCR2 with high affinity. Like all chemokine receptors, CCR2 is a GPCR(Dawson 2003). CCR2 seems to be expressed in two slightly differentforms due to alternative splicing of the mRNA encoding thecarboxyterminal region, CCR2a and CCR2b (Charo 1994). These receptorsare expressed in monocytes, myeloid precursor cells and activated Tcells (Myers 1995; Qin 1996). The dissociation constant of MCP-1 to thereceptor transfected into HEK-293 cells is 260 pM which is in agreementwith values measured on monoytes (Myers 1995; Van Riper 1993).Activation of CCR2b on transfected HEK-293 cells with MCP-1 inhibitsadenylyl cyclase at a concentration of 90 pM, and mobilizesintracellular calcium at slightly higher concentrations, seeminglyindependent of phosphatidyl inositol hydrolysis. The effects on adenylylcyclase and intracellular calcium release are strongly inhibited bypertussis toxin, implying the involvement of G, type heterotrimericG-proteins in signal transduction (Myers 1995).

MCP-1 is involved in monocyte recruitment into inflamed tissues. There,resident macrophages release chemokines such as MCP-1 and others, andcytokines like TNF, IL-1β and others, which activate endothelial cellsto express a battery of adhesion molecules. The resulting “sticky”endothelium causes monocytes in the blood vessel to roll along itssurface. Here, the monocytes encounter MCP-1 presented on theendothelial surface, which binds to CCR2 on monocytes and activatesthem. This finally leads to firm arrest, spreading of monocytes alongthe endothelium, and transmigration into the surrounding tissue, wherethe monocytes differentiate into macrophages and migrate towards thesite of maximal MCP-1 concentration.

MCP-1 is a member of the chemokine family which is a family of small(ca. 8-14 kDa) heparin-binding, mostly basic and structurally relatedmolecules. They are formed predominantly in inflamed tissues andregulate the recruitment, activation, and proliferation of white bloodcells (leukocytes) (Baggiolini 1994; Springer 1995; Schall 1994).Chemokines selectively induce chemotaxis of neutrophils, eosinophils,basophils, monocytes, macrophages, mast cells, T and B cells. Inaddition to their chemotactic effect, they can selectively exert othereffects in responsive cells like changes in cell shape, transientincrease in the concentration of free intracellular calcium ions,degranulation, upregulation of integrins, formation of bioactive lipidssuch as leukotrienes, prostaglandins, thromboxans, or respiratory burst(release of reactive oxygen species for destruction of pathogenicorganisms or tumor cells). Thus, by provoking the release of furtherproinflammatory mediators, chemotaxis and extravasation of leukocytestowards sites of infection or inflammation, chemokines triggerescalation of the inflammatory response.

Based on the arrangement of the first two of four conserved cysteinresidues, the chemokines are divided into four classes: CC orβ-chemokines in which the cysteins are in tandem, CXC or α-chemokines,where they are separated by one additional amino acid residue, XC or γchemokines with lymphotactin as only representant to date, that possessonly one disulfide bridge, and CX3C-chemokines which feature three aminoacid residues between the cysteins, with membrane-bound fractalkin asonly class member known to date (Bazan 1997).

The CXC chemokines act primarily on neutrophils, in particular those CXCchemokines that carry the amino acid sequence ELR on their aminoterminus. Examples of CXC chemokines that are active on neutrophils areIL-8, GROα, -β, and -γ, NAP-2, ENA-78 and GCP-2. The CC chemokines acton a larger variety of leukocytes, such as monocytes, macrophages,eosinophils, basophils, as well as T and B lymphocytes (Oppenheim 1991;Baggiolini 1994; Miller 1992; Jose 1994; Ponath 1996a). Examples ofthese are 1-309; MCP-1, -2, -3, -4, MIP-1α and -β, RANTES, and eotaxin.

Chemokines act through receptors that belong to a superfamily of seventransmembrane-spanning G protein-coupled receptors (GPCRs; Murphy2000)). Generally speaking, chemokine and chemokine receptorinteractions tend to be promiscuous in that one chemokine can bind manychemokine receptors and conversely a single chemokine receptor caninteract with several chemokines. Some known receptors for the CCchemokines include CCR1, which binds MIP-1α and RANTES (Neote 1993; Gao1993); CCR2, which binds chemokines including MCP-1, -2, -3, and -4(Charo 1994; Myers 1995; Gong 1997; Garcia-Zepeda 1996); CCR3, whichbinds chemokines including eotaxin, RANTES, and MCP-3 (Ponath 1996b);CCR4, which has been found to signal in response to MCP-1, MIP-1α, andRANTES (Power 1995); and CCR5, which has been shown to signal inresponse to MIP-1α and -β, and RANTES (Boring 1996; Raport 1996; Samson1996).

As mentioned above, all four members of the MCP family (1-4) bind toCCR2, whereas MCP-2, MCP-3, and MCP-4 can also interact with CCR1 andCCR3 (Gong 1997; Heath 1997; Uguccioni 1997) and, in the case of MCP-2.CCR5 (Ruffing 1998). Another CC chemokine showing high homology with theMCP family is eotaxin, which was originally isolated from thebronchoalveolar lavage fluid taken from allergen-challenged, sensitizedguinea pigs (Jose 1994). It has been shown that eotaxin is also able toactivate CCR2 (Martinelli 2001).

The problem underlying the present invention is to provide a means whichspecifically interacts with MCP-1. More specifically, the problemunderlying the present invention is to provide for a nucleic acid basedmeans which specifically interacts with MCP-1.

A further problem underlying the present invention is to provide a meansfor the manufacture of a medicament for the treatment of a human ornon-human diseases, whereby the disease is characterized by MCP-1 beingeither directly or indirectly involved in the pathogenetic mechanism ofsuch disease.

A still further problem underlying the present invention is to provide ameans for the manufacture of a diagnostic agent for the treatment of adisease, whereby the disease is characterized by MCP-1 being eitherdirectly or indirectly involved in the pathogenetic mechanism of suchdisease.

These and other problems underlying the present invention are solved bythe subject matter of the attached independent claims. Preferredembodiments may be taken from the dependent claims.

The problem underlying the present invention is also solved in a firstaspect by a nucleic acid, preferably binding to MCP-1, selected from thegroup comprising type 1A nucleic acids, type 1B nucleic acids, type 2nucleic acids, type 3 nucleic acids, type 4 nucleic acids and nucleicacids having a nucleic acid sequence according to any of SEQ. ID. No. 87to 115.

In a first subaspect of the first aspect the type 1A nucleic acidcomprises in 5′->3′ direction a first stretch Box B1A, a second stretchBox B2, a third stretch Box B3, a fourth stretch Box B4, a fifth stretchBox B5, a sixth stretch Box B6 and a seventh stretch Box B1B, whereby

-   -   the first stretch Box B1A and the seventh stretch Box B1B        optionally hybridize with each other, whereby upon hybridization        a double-stranded structure is formed,    -   the first stretch Box B1A comprises a nucleotide sequence of        AGCRUG,    -   the second stretch Box B2 comprises a nucleotide sequence of        CCCGGW,    -   the third stretch Box B3 comprises a nucleotide sequence of GUR,    -   the fourth stretch Box B4 comprises a nucleotide sequence of        RYA,    -   the fifth stretch Box B5 comprises a nucleotide sequence of        GGGGGRCGCGAYC    -   the sixth stretch Box B6 comprises a nucleotide sequence of        UGCAAUAAUG or URYAWUUG, and    -   the seventh stretch Box B1B comprises a nucleotide sequence of        CRYGCU.

In a preferred embodiment of the first subaspect

-   -   the first stretch Box B1A comprises a nucleotide sequence of        AGCGUG.

In an embodiment of the first subaspect

-   -   the second stretch Box B2 comprises a nucleotide sequence of        CCCGGU.

In an embodiment of the first subaspect

-   -   the third stretch Box B3 comprises a nucleotide sequence of GUG.

In an embodiment of the first subaspect

-   -   the fourth stretch Box B4 comprises a nucleotide sequence of        GUA.

In an embodiment of the first subaspect

-   -   the fifth stretch Box B5 comprises a nucleotide sequence of        GGGGGGCGCGACC.

In an embodiment of the first subaspect

-   -   the sixth stretch Box B6 comprises a nucleotide sequence of        UACAUUUG.

In an embodiment of the first subaspect

-   -   the seventh stretch Box B1B comprises a nucleotide sequence of        CACGCU.

In an embodiment of the first subaspect the nucleic acid comprises anucleic acid sequence according to SEQ. ID. No 21.

In a second subaspect of the first aspect the type 1B nucleic acidcomprises in 5′->3′ direction a first stretch Box B1A, a second stretchBox B2, a third stretch Box B3, a fourth stretch Box B4, a fifth stretchBox B5, a sixth stretch Box B6 and a seventh stretch Box B1B, whereby

-   -   the first stretch Box B1A and the seventh stretch Box B1B        optionally hybridize with each other, whereby upon hybridization        a double-stranded structure is formed,    -   the first stretch Box B1A comprises a nucleotide sequence of        AGYRUG,    -   the second stretch Box B2 comprises a nucleotide sequence of        CCACGCU or CCAGY,    -   the third stretch Box B3 comprises a nucleotide sequence of GUG,    -   the fourth stretch Box B4 comprises a nucleotide sequence of        AUG.    -   the fifth stretch Box B5 comprises a nucleotide sequence of        GGGGGGCGCGACC    -   the sixth stretch Box B6 comprises a nucleotide sequence of        CAUUUUA or CAUUUA, and    -   the seventh stretch Box B1B comprises a nucleotide sequence of        CAYRCU.

In an embodiment of the second subaspect

-   -   the first stretch Box B1A comprises a nucleotide sequence of        AGCGUG.

In an embodiment of the second subaspect

-   -   the second stretch Box B2 comprises a nucleotide sequence of        CCAGU.

In an embodiment of the second subaspect

-   -   the sixth stretch Box B6 comprises a nucleotide sequence of        CAUUUUA.

In an embodiment of the second subaspect

-   -   the seventh stretch Box B1B comprises a nucleotide sequence of        CACGCU.

In an embodiment of the second subaspect the nucleic acid comprises anucleic acid sequence according to SEQ. ID. No 28 and SEQ. ID. No 27.

In a third subaspect of the first aspect the type 2 nucleic acidcomprises in 5′->3′ direction a first stretch Box B1A, a second stretchBox B2, and a third stretch Box B1B, whereby

-   -   the first stretch Box B1A and the third stretch Box B1B        optionally hybridize with each other, whereby upon hybridization        a double-stranded structure is formed,    -   the first stretch Box B1A comprises a nucleotide sequence        selected from the group comprising ACGCA, CGCA and GCA,    -   the second stretch Box B2 comprises a nucleotide sequence of        CSUCCCUCACCGGUGCAAGUGAAGCCGYGGCUC, and    -   the third stretch Box B1B comprises a nucleotide sequence        selected from the group comprising UGCGU, UGCG and UGC.

In an embodiment of the third subaspect

-   -   the second stretch Box B2 comprises a nucleotide sequence of        CGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUC.

In an embodiment of the third subaspect

-   -   a) the first stretch Box B1A comprises a nucleotide sequence of        ACGCA, and        -   the third stretch Box B1B comprises a nucleotide sequence of            UGCGU; or    -   b) the first stretch Box B1A comprises a nucleotide sequence of        CGCA, and        -   the third stretch Box B1B comprises a nucleotide sequence of            UGCG; or    -   c) the first stretch Box B1A comprises a nucleotide sequence of        GCA, and        -   the third stretch Box B1B comprises a nucleotide sequence of            UGC or UGCG.

In an embodiment of the third subaspect

-   -   the first stretch Box B1A comprises a nucleotide sequence of        GCA.

In a preferred embodiment of the third subaspect

-   -   the third stretch Box B1B comprises a nucleotide sequence of        UGCG.

In an embodiment of the third subaspect the nucleic acid comprises anucleic acid sequence according to SEQ. ID. No 37., SEQ. ID. No 116,SEQ. ID. No 117 and SEQ. ID. No 278.

In a fourth subaspect of the first aspect the type 3 nucleic acidcomprises in 5′->3′ direction a first stretch Box B1A, a second stretchBox B2A, a third stretch Box B3, a fourth stretch Box B2B, a fifthstretch Box B4, a sixth stretch Box B5A, a seventh stretch Box B, aneighth stretch Box B5B and a ninth stretch Box B1B, whereby

-   -   the first stretch Box B1A and the ninth stretch Box B1B        optionally hybridize with each other, whereby upon hybridization        a double-stranded structure is formed,    -   the second stretch Box B2A and the fourth Box B2B optionally        hybridize with each other, whereby upon hybridization a        double-stranded structure is formed,    -   the sixth stretch Box B5A and the eighth Box B5B optionally        hybridize with each other, whereby upon hybridization a        double-stranded structure is formed,    -   the first stretch Box B1A comprises a nucleotide sequence which        is selected from the group comprising GURCUGC, GKSYGC, KBBSC and        BNGC,    -   the second stretch Box B2A comprises a nucleotide sequence of        GKMGU,    -   the third stretch Box B3 comprises a nucleotide sequence of        KRRAR,    -   the fourth stretch Box B2B comprises a nucleotide sequence of        ACKMC,    -   the fifth stretch Box B4 comprises a nucleotide sequence        selected from the group comprising CURYGA, CUWAUGA, CWRMGACW and        UGCCAGUG,    -   the sixth stretch Box B5A comprises a nucleotide sequence        selected from the group comprising GGY and CWGC,    -   the seventh stretch Box B6 comprises a nucleotide sequence        selected from the group comprising YAGA, CKAAU and CCUUUAU,    -   the eighth stretch Box B5B comprises a nucleotide sequence        selected from the group comprising GCYR and GCWG, and    -   the ninth stretch Box B1B comprises a nucleotide sequence        selected from the group comprising GCAGCAC, GCRSMC, GSVVM and        GCNV.

In an embodiment of the fourth subaspect

-   -   the third stretch Box B3 comprises a nucleotide sequence of        GAGAA or UAAAA

In an embodiment of the fourth subaspect

-   -   the fifth stretch Box B4 comprises a nucleotide sequence of        CAGCGACU or CAACGACU.

In an embodiment of the fourth subaspect

-   -   the fifth stretch Box B4 comprises a nucleotide sequence of        CAGCGACU and Box B3 comprises a nucleotide sequence of UAAAA.

In an embodiment of the fourth subaspect

-   -   the fifth stretch Box B4 comprises a nucleotide sequence of        CAACGACU and Box B3 comprises a nucleotide sequence of GAGAA.

In an embodiment of the fourth subaspect

-   -   the seventh stretch Box B6 comprises a nucleotide sequence of        UAGA.

In an embodiment of the fourth subaspect

-   -   a) the first stretch Box B1A comprises a nucleotide sequence of        GURCUGC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GCAGCAC; or    -   b) the first stretch Box B1A comprises a nucleotide sequence of        GKSYGC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GCRSMC; or    -   c) the first stretch Box B1A comprises a nucleotide sequence of        KBBSC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GSVVM; or    -   d) the first stretch Box B1A comprises a nucleotide sequence of        BNGC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GCNV.

In a preferred embodiment of the fourth subaspect

-   -   a) the first stretch Box B1A comprises a nucleotide sequence of        GUGCUGC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GCAGCAC; or    -   b) the first stretch Box B1A comprises a nucleotide sequence of        GUGCGC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GCGCAC; or    -   c) the first stretch Box B1A comprises a nucleotide sequence of        KKSSC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GSSMM, or    -   d) the first stretch Box B1A comprises a nucleotide sequence of        SNGC, and        -   the ninth stretch Box B1B comprises a nucleotide sequence of            GCNS.

In a further preferred embodiment of the fourth subaspect

-   -   the first stretch Box B1A comprises a nucleotide sequence of        GGGC, and    -   the ninth stretch Box B1B comprises a nucleotide sequence of        GCCC.

In an embodiment of the fourth subaspect the second stretch Box B2Acomprises a nucleotide sequence of GKMGU and the fourth stretch Box B2Bcomprises a nucleotide sequence of ACKMC.

In a preferred embodiment of the fourth subaspect the second stretch BoxB2A comprises a nucleotide sequence of GUAGU and the fourth stretch BoxB2B comprises a nucleotide sequence of ACUAC.

In an embodiment of the fourth subaspect

-   -   a) the sixth stretch Box B5A comprises a nucleotide sequence of        GGY, and        -   the eighth stretch Box B5B comprises a nucleotide sequence            of GCYR; or    -   b) the sixth stretch Box B5A comprises a nucleotide sequence of        CWGC, and        -   the eighth stretch Box B5B comprises a nucleotide sequence            of GCWG.

In a preferred embodiment of the fourth subaspect

-   -   the sixth stretch Box B5A comprises a nucleotide sequence of        GGC, and    -   the eighth stretch Box B5B comprises a nucleotide sequence of        GCCG.

In a more preferred embodiment of the fourth subaspect the sixth stretchBox B5A hybridizes with the nucleotides GCY of the eighth stretch BoxB5B.

In an embodiment of the fourth subaspect the nucleic acid comprises anucleic acid sequence according to SEQ. ID. No 56.

In an embodiment of the fourth subaspect the nucleic acid comprises anucleic acid sequence selected from the group comprising the nucleicacid sequences according to SEQ. ID. No 57 to 61, SEQ. ID. No 67 to 71and SEQ. ID. No 73.

In a fifth subaspect of the first aspect the type 4 nucleic acidcomprises in 5′->3′ direction a first stretch Box B1A, a second stretchBox B2, a third stretch Box B1B whereby

-   -   the first stretch Box B1A and the third stretch Box B1B        optionally hybridize with each other, whereby upon hybridization        a double-stranded structure is formed,    -   the first stretch Box B1A comprises a nucleotide sequence        selected from the group comprising AGCGUGDU, GCGCGAG, CSKSUU,        GUGUU, and UGUU;    -   the second stretch Box B2 comprises a nucleotide sequence        selected from the group comprising AGNDRDGBKGGURGYARGUAAAG,        AGGUGGGUGGUAGUAAGUAAAG and CAGGUGGGUGGUAGAAUGUAAAGA, and    -   the third stretch Box B1B comprises a nucleotide sequence        selected from the group comprising GNCASGCU, CUCGCGUC, GRSMSG,        GRCAC, and GGCA.

In an embodiment of the fifth subaspect

-   -   a) the first stretch Box B1A comprises a nucleotide sequence of        GUGUU, and        -   the third stretch Box B1B comprises a nucleotide sequence of            GRCAC;    -   b) the first stretch Box B1A comprises a nucleotide sequence of        GCGCGAG. and        -   the third stretch Box B1B comprises a nucleotide sequence of            CUCGCGUC; or    -   c) the first stretch Box B1A comprises a nucleotide sequence of        CSKSUU, and        -   the third stretch Box B1B comprises a nucleotide sequence of            GRSMSG, or    -   d) the first stretch Box B1A comprises a nucleotide sequence of        UGUU, and        -   the third stretch Box B1B comprises a nucleotide sequence of            GGCA, or    -   e) the first stretch Box B1A comprises a nucleotide sequence of        AGCGUGDU, and        -   the third stretch Box B1B comprises a nucleotide sequence of            GNCASGCU.

In a preferred embodiment of the fifth subaspect the first stretch BoxB1A comprises a nucleotide sequence of CSKSUU and the third stretch BoxB1B comprises a nucleotide sequence of GRSMSG.

In a more preferred embodiment of the fifth subaspect the first stretchBox B1A comprises a nucleotide sequence of CCGCUU and the third stretchBox B1B comprises a nucleotide sequence of GGGCGG.

In an embodiment of the fifth subaspect

-   -   the second stretch Box B2 comprises a nucleotide sequence of        AGGUGGGUGGUAGUAAGUAAAG.

In an embodiment of the fifth subaspect the nucleic acid comprises anucleic acid sequence according to SEQ. ID. No 80.

In an embodiment of the first to the fifth subaspect the nucleic acid iscapable of binding MCP-1, preferably human MCP-1.

In an embodiment of the first to the fifth subaspect the nucleic acid iscapable of binding a chemokine, whereby the chemokine is selected fromthe group comprising eotaxin, MCP-1. MCP-2 and MCP-3.

In an embodiment of the first to the fifth subaspect the nucleic acid iscapable of binding a chemokine, whereby the chemokine is selected fromthe group comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the first to the fifth subaspect the nucleic acid iscapable of binding MCP-1, whereby MCP-1 is preferably selected from thegroup comprising monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1,canine MCP-1, porcine MCP-1 and human MCP-1.

In an embodiment of the first to the fifth subaspect the nucleic acid iscapable of binding human MCP-1.

In a preferred embodiment of the first to the fifth subaspect the MCP-Lhas an amino acid sequence according to SEQ ID No. 1.

The problem underlying the present invention is solved in a secondaspect by a nucleic acid, preferably binding to murine MCP-1, wherebythe nucleic acid comprises a nucleic acid sequence according to SEQ. ID.No. 122, SEQ. ID. No. 253 and SEQ. ID. No. 254.

The problem underlying the present invention is solved in a third aspectby a nucleic acid, preferably binding to murine MCP-1, whereby thenucleic acid comprises a nucleic acid sequence according to SEQ. ID. No.127.

In an embodiment of the second and third aspect the murine MCP-1comprises an amino acid sequence according to SEQ ID No. 2.

In an embodiment of the first to the third aspect the nucleic acidcomprises a modification, whereby the modification is preferably a highmolecular weight moiety and/or whereby the modification preferablyallows to modify the characteristics of the nucleic acid according toany of the first, second and third aspect in terms of residence time inthe animal or human body, preferably the human body.

In a preferred embodiment of the first to the third aspect themodification is selected from the group comprising a HES moiety and aPEG moiety.

In a more preferred embodiment of the first to the third aspect themodification is a PEG moiety consisting of a straight or branched PEG,whereby the molecular weight of the PEG moiety is preferably from about20 to 120 kD, more preferably from about 30 to 80 kD and most preferablyabout 40 kD.

In an alternative more preferred embodiment of the first to the thirdaspect the modification is a HES moiety, whereby preferably themolecular weight of the HES moiety is from about 10 to 130 kD, morepreferably from about 30 to 130 kD and most preferably about 100 kD.

In an embodiment of the first to the third aspect the modification iscoupled to the nucleic acid via a linker.

In an embodiment of the first to the third aspect the modification iscoupled to the nucleic acid at its 5′-terminal nucleotide and/or its3′-terminal nucleotide and/or to a nucleotide of the nucleic acidbetween the 5′-terminal nucleotide and the 3′-terminal nucleotide.

In an embodiment of the first to the third aspect the nucleotides of orthe nucleotides forming the nucleic acid are L-nucleotides.

In an embodiment of the first to the third aspect the nucleic acid is anL-nucleic acid.

In an embodiment of the first to the third aspect the moiety of thenucleic acid capable of binding MCP-1 consists of L-nucleotides.

The problem underlying the present invention is solved in a fourthaspect by a pharmaceutical composition comprising a nucleic acidaccording to the first, second and third aspect and optionally a furtherconstituent, whereby the further constituent is selected from the groupcomprising pharmaceutically acceptable excipients, pharmaceuticallyacceptable carriers and pharmaceutically active agents.

In an embodiment of the fourth aspect the pharmaceutical compositioncomprises a nucleic acid according to any of the first to third aspectand a pharmaceutically acceptable carrier.

The problem underlying the present invention is solved in a fifth aspectby the use of a nucleic acid according to the first, second and thirdaspect for the manufacture of a medicament.

In an embodiment of the fifth aspect the medicament is for use in humanmedicine or for use in veterinary medicine.

The problem underlying the present invention is solved in a sixth aspectby the use of a nucleic acid according to the first, second and thirdaspect for the manufacture of a diagnostic means.

In an embodiment of the fifth aspect and in an embodiment of the sixthaspect the medicament and diagnostic means, respectively, is for thetreatment and/or prevention and diagnosis, respectively, of a disease ordisorder selected from the group comprising inflammatory diseases,autoimmune diseases, autoimmune encephalomyelitis, stroke, acute andchronic multiple sclerosis, chronic inflammation, rheumatoid arthritis,renal diseases, restenosis, restenosis after angioplasty, acute andchronic allergic reactions, primary and secondary immunologic orallergic reactions, asthma, conjunctivitis, bronchitis, cancer,atherosclerosis, arteriosclerotic cardiovasular heart failure or stroke,psoriasis, psoriatic arthritis, inflammation of the nervous system,atopic dermatitis, colitis, endometriosis, uveitis, retinal disordersincluding macular degeneration, retinal detachment, diabeticretinopathy, retinopathy of prematurity, retinitis pigmentosa,proliferative vitreoretinopathy, and central serous chorioretinopathy,idiopathic pulmonary fibrosis, sarcoidosis, polymyositis,dermatomyositis, avoidance of immunosuppression, reducing the risk ofinfection, sepsis, renal inflammation, glomerulonephritis, rapidprogressive glomerulonephritis, proliferative glomerulonephritis,diabetic nephropathy, obstructive nephropathy, acute tubular necrosis,and diffuse glomerulosclerosis, systemic lupus erythematosus, chronicbronchitis, Behçet's disease, amyotrophic lateral sclerosis (ALS),premature atherosclerosis after Kawasaki's disease, myocardialinfarction, obesity, chronic liver disease, peyronie's disease, acutespinal chord injury, lung or kidney transplantation, myocarditis,Alzheimer's disease and neuropathy, breast carcinoma, gastric carcinoma,bladder cancer, ovarian cancer, hamartoma, colorectal carcinoma, colonicadenoma, pancreatitis, chronic obstructive pulmonary disease (COPD) andinflammatory bowel diseases such as Crohn's disease or ulcerativecolitis.

Without wishing to be bound be any theory, the suitability of thenucleic acids of the present invention for diagnostic purposes is mostlybased on an increased or decreased chemokine level, whereby suchchemokine is selected from the group comprising eotaxin, MCP-1, MCP-2and MCP-3, more specifically MCP-1. It will be acknowledged by theperson skilled in the art that most of the aforementioned diseases showsuch increased or decreased chemokine level.

The problem underlying the present invention is solved in a seventhaspect by a complex comprising a chemokine and a nucleic acid accordingto the first, second and third aspect, whereby the chemokine is selectedfrom the group comprising eotaxin, MCP-1, MCP-2 and MCP-3, wherebypreferably the complex is a crystalline complex.

In an embodiment of the seventh aspect the chemokine is selected fromthe group comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the seventh aspect the chemokine is MCP-1, wherebyMCP-1 is preferably selected from the group comprising human MCP-1,monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1, canine MCP-1 andporcine MCP-1, more preferably MCP-1 is human MCP-1.

The problem underlying the present invention is solved in an eighthaspect by the use of a nucleic acid according to the first, second andthird aspect for the detection of a chemokine, whereby the chemokine isselected from the group comprising eotaxin, MCP-1, MCP-2 and MCP-3.

In an embodiment of the eighth aspect the chemokine is selected from thegroup comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the eighth aspect the chemokine is MCP-1, wherebyMCP-1 is preferably selected from the group comprising human MCP-1,monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1, canine MCP-1 andporcine MCP-1, more preferably MCP-1 is human MCP-1.

The problem underlying the present invention is solved in a ninth aspectby a method for the screening of a chemokine antagonist or a chemokineagonist comprising the following steps:

-   -   providing a candidate chemokine antagonist and/or a candidate        chemokine agonist.    -   providing a nucleic acid according to the first, second or third        aspect,    -   providing a test system which provides a signal in the presence        of a chemokine antagonist and/or a chemokine agonist, and    -   determining whether the candidate chemokine antagonist is a        chemokine antagonist and/or whether the candidate chemokine        agonist is a chemokine agonist,        whereby the chemokine is selected from the group comprising        eotaxin, MCP-1, MCP-2 and MCP-3.

In an embodiment of the nineth aspect the chemokine is selected from thegroup comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the nineth aspect the chemokine is MCP-1, wherebyMCP-1 is preferably selected from the group comprising human MCP-1,monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1, canine MCP-1 andporcine MCP-1, more preferably MCP-1 is human MCP-1.

The problem underlying the present invention is solved in a tenth aspectby a method for the screening of a chemokine agonist and/or a chemokineantagonist comprising the following steps:

-   -   providing a chemokine immobilised to a phase, preferably a solid        phase,    -   providing a nucleic acid according to the first, second or third        aspect, preferably a nucleic acid according to the first aspect        which is labelled,    -   adding a candidate chemokine agonist and/or a candidate        chemokine antagonist, and    -   determining whether the candidate chemokine agonist is a        chemokine agonist and/or whether the candidate chemokine        antagonist is a chemokine antagonist,        whereby the chemokine is selected from the group comprising        eotaxin, MCP-1, MCP-2 and MCP-3.

In an embodiment of the tenth aspect the determining is carried out suchthat it is assessed whether the nucleic acid is replaced by thecandidate chemokine_agonist or by a candidate chemokine antagonist.

In an embodiment of the tenth aspect the chemokine is selected from thegroup comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the tenth aspect the chemokine is MCP-1, wherebyMCP-1 is preferably selected from the group comprising human MCP-1,monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1, canine MCP-L andporcine MCP-1, more preferably MCP-1 is human MCP-1.

The problem underlying the present invention is solved in an eleventhaspect by a kit for the detection of a chemokine, comprising a nucleicacid according to the first, second and third aspect, whereby thechemokine is selected from the group comprising eotaxin, MCP-1, MCP-2and MCP-3.

In an embodiment of the eleventh aspect the chemokine is selected fromthe group comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the eleventh aspect the chemokine is MCP-1, wherebyMCP-1 is preferably selected from the group comprising human MCP-1,monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1, canine MCP-1 andporcine MCP-1, more preferably MCP-1 is human MCP-t.

The problem underlying the present invention is solved in a twelfthaspect by a chemokine antagonist obtainable by the method according tothe tenth aspect or the ninth aspect, whereby the chemokine is selectedfrom the group comprising eotaxin, MCP-1, MCP-2 and MCP-3.

In an embodiment of the twelfth aspect the chemokine is selected fromthe group comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the twelfth aspect the chemokine is MCP-1, wherebyMCP-1 is preferably selected from the group comprising human MCP-1,monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1, canine MCP-1 andporcine MCP-1, more preferably MCP-1 is human MCP-1.

The problem underlying the present invention is solved in a thirteenthaspect by a chemokine agonist obtainable by the method according to thetenth aspect or the ninth aspect, whereby the chemokine is selected fromthe group comprising eotaxin, MCP-1, MCP-2 and MCP-3.

In an embodiment of the thirteenth aspect the chemokine is selected fromthe group comprising human eotaxin, human MCP-1, human MCP-2 and humanMCP-3.

In an embodiment of the thirteenth aspect the chemokine is MCP-1,whereby MCP-1 is preferably selected from the group comprising humanMCP-1, monkey MCP-1, horse MCP-1, rabbit MCP-1, bovine MCP-1, canineMCP-1 and porcine MCP-1, more preferably MCP-1 is human MCP-1.

It will be acknowledged by the person skilled in the art that achemokine agonist and/or a chemokine antagonist is preferably an agonistand antagonist, respectively, addressing the respective chemokine asspecified herein. Accordingly, the chemokine agonist and chemokineantagonist is, for example, an MCP-1 agonist and MCP-1 antagonist,respectively.

The problem underlying the present invention is solved in a fourteenthaspect by a method for the detection of the nucleic acid according toany of the first, second and third aspect in a sample, whereby themethod comprises the steps of:

-   -   a) providing a sample containing the nucleic acid according to        the present invention;    -   b) providing a capture probe, whereby the capture probe is at        least partially complementary to a first part of the nucleic        acid according to any of the first, second and third aspect, and        a detection probe, whereby the detection probe is at least        partially complementary to a second part of the nucleic acid        according to any of the first, second and third aspect, or,        alternatively, the capture probe is at least partially        complementary to a second part of the nucleic acid according to        any of the first, second and third aspect and the detection        probe is at least partially complementary to the first part of        the nucleic acid according to any of the first, second and third        aspect,    -   c) allowing the capture probe and the detection probe to react        either simultaneously or in any order sequentially with the        nucleic acid according to any of the first, second and third        aspect or part thereof;    -   d) optionally detecting whether or not the capture probe is        hybridized to the nucleic acid according to the nucleic acid        according to any of the first, second and third aspect provided        in step a); and    -   e) detecting the complex formed in step c) consisting of the        nucleic acid according to any of the first, second and third        aspect, and the capture probe and the detection probe.

In an embodiment of the fourteenth aspect the detection probe comprisesa detection means, and/or whereby the capture probe can be immobilizedto a support, preferably a solid support.

In an embodiment of the fourteenth aspect any detection probe which isnot part of the complex is removed from the reaction so that in step e)only a detection probe which is part of the complex, is detected.

In an embodiment of the fourteenth aspect step e) comprises the step ofcomparing the signal generated by the detection means when the captureprobe and the detection probe are hybridized in the presence of thenucleic acid according to any of the first, second or third aspect orpart thereof, and in the absence of said nucleic acid or part thereof.

In an embodiment of the fourteenth aspect the nucleic acid to bedetected is the nucleic acid having a nucleic acid sequence according toSEQ. ID. NOs. 37, 116, 117 or 278, and the capture probe or detectionprobe comprises a nucleic acid sequence according to SEQ. ID. NO. 255 orSEQ. ID. NO. 256.

In an embodiment of the fourteenth aspect the nucleic acid to bedetected is the nucleic acid having a nucleic acid sequence according toSEQ. ID. NOs. 122, 253 or 254 and the capture probe or detection probecomprises a nucleic acid sequence according to SEQ. ID. NO. 281 and SEQ.ID. NO. 282.

The problem underlying the present invention is also solved by thesubject matter of the independent claims attached hereto. Preferredembodiment may be taken from the attached dependent claims.

The features of the nucleic acid according to the present invention asdescribed herein can be realised in any aspect of the present inventionwhere the nucleic acid is used, either alone or in any combination.

Human as well as murine MCP-1 are basic proteins having the amino acidsequence according to SEQ. ID. Nos. 1 and 2, respectively.

The finding that short high affinity binding nucleic acids to MCP-1could be identified, is insofar surprising as Eaton et al. (1997)observed that the generation of aptamers, i.e. D-nucleic acids bindingto a target molecule, directed to a basic protein is in general verydifficult because this kind of target produces a high but non-specificsignal-to-noise ratio. This high signal-to-noise ratio results from thehigh non-specific affinity shown by nucleic acids for basic targets suchas MCP-1.

As outlined in more detail in the claims and example 1, the presentinventors could more surprisingly identify a number of different MCP-1binding nucleic acid molecules, whereby most of the nucleic acids couldbe characterised in terms of stretches of nucleotide which are alsoreferred to herein as Boxes. The various MCP-1 binding nucleic acidmolecules can be categorised based on said Boxes and some structuralfeatures and elements, respectively. The various categories thus definedare also referred to herein as types and more specifically as type 1A,type 1B, type 2, type 3 and type 4.

The nucleic acids according to the present invention shall also comprisenucleic acids which are essentially homologous to the particularsequences disclosed herein. The term substantially homologous shall beunderstood such that the homology is at least 75%, preferably 85%, morepreferably 90% and most preferably more than 95%, 96%, 97%, 98% or 99%.

The actual percentage of homologous nucleotides present in the nucleicacid according to the present invention will depend on the total numberof nucleotides present in the nucleic acid. The percent modification canbe based upon the total number of nucleotides present in the nucleicacid.

The homology can be determined as known to the person skilled in theart. More specifically, a sequence comparison algorithm then calculatesthe percent sequence identity for the test sequence(s) relative to thereference sequence, based on the designated program parameters. The testsequence is preferably the sequence or nucleic acid molecule which issaid to be or to be tested whether it is homologous, and if so, to whatextent, to another nucleic acid molecule, whereby such another nucleicacid molecule is also referred to as the reference sequence. In anembodiment, the reference sequence is a nucleic acid molecule asdescribed herein, more preferably a nucleic acid molecule having asequence according to any of SEQ. ID. NOs. 10 to 129, 132 to 256 and278-282. Optimal alignment of sequences for comparison can be conducted,e.g., by the local homology algorithm of Smith & Waterman (Smith &Waterman, 1981) by the homology alignment algorithm of Needleman &Wunsch (Needleman & Wunsch, 1970) by the search for similarity method ofPearson & Lipman (Pearson & Lipman, 1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection.

One example of an algorithm that is suitable for determining percentsequence identity is the algorithm used in the basic local alignmentsearch tool (hereinafter “BLAST”), see, e.g. Altschul et al (Altschul etal. 1990 and Altschul et al, 1997). Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (hereinafter “NCBI”). The default parametersused in determining sequence identity using the software available fromNCBI, e.g., BLASTN (for nucleotide sequences) and BLASTP (for amino acidsequences) are described in McGinnis et al (McGinnis et al, 2004).

The term inventive nucleic acid or nucleic acid according to the presentinvention shall also comprise those nucleic acids comprising the nucleicacids sequences disclosed herein or part thereof, preferably to theextent that the nucleic acids or said parts are involved in the bindingto MCP-1. The term inventive nucleic acid as preferably used herein,shall also comprise in an embodiment a nucleic acid which is suitable tobind to any molecule selected from the group comprising MCP-2, MCP-3,MCP-4, and eotaxin. It will be acknowledged by the ones skilled in theart that the individual nucleic acids according to the present inventionwill bind to one or several of such molecules. Such nucleic acid is, inan embodiment, one of the nucleic acid molecules described herein, or aderivative and/or a metabolite thereof, whereby such derivative and/ormetabolite are preferably a truncated nucleic acid compared to thenucleic acid molecules described herein. Truncation may be related toeither or both of the ends of the nucleic acids as disclosed herein.Also, truncation may be related to the inner sequence of nucleotides ofthe nucleic acid. i.e. it may be related to the nucleotide(s) betweenthe 5′ and the 3′ terminal nucleotide, respectively. Moreover,truncation shall comprise the deletion of as little as a singlenucleotide from the sequence of the nucleic acids disclosed herein.Truncation may also be related to more than one stretch of the inventivenucleic acid(s), whereby the stretch can be as little as one nucleotidelong. The binding of a nucleic acid according to the present invention,preferably to a molecule selected from the group comprising MCP-1,MCP-2, MCP-3, MCP-4 and eotaxin, can be determined by the ones skilledin the art using routine experiments or by using or adopting a method asdescribed herein, preferably as described herein in the example part. Itis within an embodiment of the present invention, unless explicitlyindicated to the contrary, that whenever it is referred herein to thebinding of the nucleic acids according to the present invention to orwith MCP-1, this applies also to the binding of the nucleic acidsaccording to the present invention to or with any molecule selected fromthe group comprising MCP-2, MCP-3, MCP-4 and eotaxin.

The nucleic acids according to the present invention may be eitherD-nucleic acids or L-nucleic acids. Preferably, the inventive nucleicacids are L-nucleic acids. In addition it is possible that one orseveral parts of the nucleic acid are present as D-nucleic acids or atleast one or several parts of the nucleic acids are L-nucleic acids. Theterm “part” of the nucleic acids shall mean as little as one nucleotide.Such nucleic acids are generally referred to herein as D- and L-nucleicacids, respectively. Therefore, in a particularly preferred embodiment,the nucleic acids according to the present invention consist ofL-nucleotides and comprise at least one D-nucleotide. Such D-nucleotideis preferably attached to a part different from the stretches definingthe nucleic acids according to the present invention, preferably thoseparts thereof, where an interaction with other parts of the nucleic acidis involved. Preferably, such D-nucleotide is attached at a terminus ofany of the stretches and of any nucleic acid according to the presentinvention, respectively. In a further preferred embodiment, suchD-nucleotides may act as a spacer or a linker, preferably attachingmodifications such as PEG and HES to the nucleic acids according to thepresent invention.

It is also within an embodiment of the present invention that each andany of the nucleic acid molecules described herein in their entirety interms of their nucleic acid sequence(s) are limited to the particularnucleotide sequence(s). In other words, the terms “comprising” or“comprise(s)” shall be interpreted in such embodiment in the meaning ofcontaining or consisting of.

It is also within the present invention that the nucleic acids accordingto the present invention are part of a longer nucleic acid whereby thislonger nucleic acid comprises several parts whereby at least one suchpart is a nucleic acid according to the present invention, or a partthereof. The other part(s) of these longer nucleic acids can be eitherone or several D-nucleic acid(s) or one or several L-nucleic acid(s).Any combination may be used in connection with the present invention.These other part(s) of the longer nucleic acid either alone or takentogether, either in their entirety or in a particular combination, canexhibit a function which is different from binding, preferably frombinding to MCP-1. One possible function is to allow interaction withother molecules, whereby such other molecules preferably are differentfrom MCP-1, such as, e.g., for immobilization, cross-linking, detectionor amplification. In a further embodiment of the present invention thenucleic acids according to the invention comprise, as individual orcombined moieties, several of the nucleic acids of the presentinvention. Such nucleic acid comprising several of the nucleic acids ofthe present invention is also encompassed by the term longer nucleicacid.

L-nucleic acids as used herein are nucleic acids consisting ofL-nucleotides, preferably consisting completely of L-nucleotides.

D-nucleic acids as used herein are nucleic acids consisting ofD-nucleotides, preferably consisting completely of D-nucleotides.

The terms nucleic acid and nucleic acid molecule are used herein in aninterchangeable manner if not explicitly indicated to the contrary.

Also, if not indicated to the contrary, any nucleotide sequence is setforth herein in 5′→3′ direction.

Irrespective of whether the inventive nucleic acid consists ofD-nucleotides, L-nucleotides or a combination of both with thecombination being e.g. a random combination or a defined sequence ofstretches consisting of at least one L-nucleotide and at least oneD-nucleic acid, the nucleic acid may consist of desoxyribonucleotide(s),ribonucleotide(s) or combinations thereof.

Designing the inventive nucleic acids as L-nucleic acid is advantageousfor several reasons. L-nucleic acids are enantiomers of naturallyoccurring nucleic acids. D-nucleic acids, however, are not very stablein aqueous solutions and particularly in biological systems orbiological samples due to the widespread presence of nucleases.Naturally occurring nucleases, particularly nucleases from animal cellsare not capable of degrading L-nucleic acids. Because of this thebiological half-life of the L-nucleic acid is significantly increased insuch a system, including the animal and human body. Due to the lackingdegradability of L-nucleic acid no nuclease degradation products aregenerated and thus no side effects arising therefrom observed. Thisaspect delimits the L-nucleic acid of factually all other compoundswhich are used in the therapy of diseases and/or disorders involving thepresence of MCP-1. L-nucleic acids which specifically bind to a targetmolecule through a mechanism different from Watson Crick base pairing,or aptamers which consists partially or completely of L-nucleotides,particularly with those parts of the aptamer being involved in thebinding of the aptamer to the target molecule, are also calledspiegelmers.

It is also within the present invention that the inventive nucleicacids, also referred to herein as nucleic acids according to theinvention, regardless whether they are present as D-nucleic acids,L-nucleic acids or D, L-nucleic acids or whether they are DNA or RNA,may be present as single-stranded or double-stranded nucleic acids.Typically, the inventive nucleic acids are single-stranded nucleic acidswhich exhibit defined secondary structures due to thdie primary sequenceand may thus also form tertiary structures. The inventive nucleic acids,however, may also be double-stranded in the meaning that two strandswhich are complementary or partially complementary to each other arehybridised to each other. This confers stability to the nucleic acidwhich, in particular, will be advantageous if the nucleic acid ispresent in the naturally occurring D-form rather than the L-form.

The inventive nucleic acids may be modified. Such modifications may berelated to the single nucleotide of the nucleic acid and are well knownin the art. Examples for such modification are described in, amongothers, Venkatesan (2003); Kusser (2000); Aurup (1994); Cummins (1995);Eaton (1995); Green (1995); Kawasaki (1993); Lesnik (1993); and Miller(1993). Such modification can be a H atom, a F atom or O—CH3 group orNH2-group at the 2′ position of the individual nucleotide of which thenucleic acid consists. Also, the nucleic acid according to the presentinvention can comprises at least one LNA nucleotide. In an embodimentthe nucleic acid according to the present invention consists of LNAnucleotides.

In an embodiment, the nucleic acids according to the present inventionmay be a multipartite nucleic acid. A multipartite nucleic acid as usedherein, is a nucleic acid which consists of at least two nucleic acidstrands. These at least two nucleic acid strands form a functional unitwhereby the functional unit is a ligand to a target molecule. The atleast two nucleic acid strands may be derived from any of the inventivenucleic acids by either cleaving the nucleic acid to generate twostrands or by synthesising one nucleic acid corresponding to a firstpart of the inventive, i.e. overall nucleic acid and another nucleicacid corresponding to the second part of the overall nucleic acid. It isto be acknowledged that both the cleavage and the synthesis may beapplied to generate a multipartite nucleic acid where there are morethan two strands as exemplified above. In other words, the at least twonucleic acid strands are typically different from two strands beingcomplementary and hybridising to each other although a certain extent ofcomplementarity between the various nucleic acid parts may exist.

Finally it is also within the present invention that a fully closed,i.e. circular structure for the nucleic acids according to the presentinvention is realized, i.e. that the nucleic acids according to thepresent invention are closed, preferably through a covalent linkage,whereby more preferably such covalent linkage is made between the 5′ endand the 3′ end of the nucleic acid sequences as disclosed herein.

The present inventors have discovered that the nucleic acids accordingto the present invention exhibit a very favourable K_(D) value range.

A possibility to determine the binding constant is the use of the socalled biacore device, which is also known to the one skilled in theart. Affinity as used herein was also measured by the use of the“pull-down assay” as described in the examples. An appropriate measurein order to express the intensity of the binding between the nucleicacid according to the target which is in the present case MCP-1, is theso-called K_(D) value which as such as well the method for itsdetermination are known to the one skilled in the art.

The nucleic acids according to the present invention are characterizedby a certain K_(D) value. Preferably, the K_(D) value shown by thenucleic acids according to the present invention is below 1 μM. A K_(D)value of about 1 μM is said to be characteristic for a non-specificbinding of a nucleic acid to a target. As will be acknowledged by theones in the art, the K_(D) value of a group of compounds such as thenucleic acids according to the present invention are within a certainrange. The above-mentioned K_(D) of about 1 μM is a preferred upperlimit for the K_(D) value. The preferred lower limit for the K_(D) oftarget binding nucleic acids can be about 10 picomolar or higher. It iswithin the present invention that the K_(D) values of individual nucleicacids binding to MCP-1 is preferably within this range. Preferred rangescan be defined by choosing any first number within this range and anysecond number within this range. Preferred upper values are 250 nM and100 nM, preferred lower values are 50 nM, 10 nM, 1 nM, 100 pM and 10 pM.

The nucleic acid molecules according to the present invention may haveany length provided that they are still able to bind to the targetmolecule. It will be acknowledged in the art that there are preferredlengths of the nucleic acids according to the present inventions.Typically, the length is between 15 and 120 nucleotides. It will beacknowledged by the ones skilled in the art that any integer between 15and 120 is a possible length for the nucleic acids according to thepresent invention. More preferred ranges for the length of the nucleicacids according to the present invention are lengths of about 20 to 100nucleotides, about 20 to 80 nucleotides, about 20 to 60 nucleotides,about 20 to 50 nucleotides and about 30 to 50 nucleotides.

It is within the present invention that the nucleic acids disclosedherein comprise a moiety which preferably is a high molecular weightmoiety and/or which preferably allows to modify the characteristics ofthe nucleic acid in terms of, among others, residence time in the animalbody, preferably the human body. A particularly preferred embodiment ofsuch modification is PEGylation and HESylation of the nucleic acidsaccording to the present invention. As used herein PEG stands forpoly(ethylene glycole) and HES for hydroxyethyl starch. PEGylation aspreferably used herein is the modification of a nucleic acid accordingto the present invention whereby such modification consists of a PEGmoiety which is attached to a nucleic acid according to the presentinvention. HESylation as preferably used herein is the modification of anucleic acid according to the present invention whereby suchmodification consists of a HES moiety which is attached to a nucleicacid according to the present invention. These modifications as well asthe process of modifying a nucleic acid using such modifications, isdescribed in European patent application EP 1 306 382, the disclosure ofwhich is herewith incorporated in its entirety by reference.

Preferably, the molecular weight of a modification consisting of orcomprising a high molecular weight moiety is about from 2,000 to 200,000Da, preferably 20,000 to 120,000 Da, particularly in case of PEG beingsuch high molecular weight moiety, and is preferably about from 3,000 to180,000 Da, more preferably from 5,000 to 130,000 Da, particularly incase of HES being such high molecular weight moiety. The process of HESmodification is, e.g., described in German patent application DE 1 2004006 249.8 the disclosure of which is herewith incorporated in itsentirety by reference.

It is within the present invention that either of PEG and HES may beused as either a linear or branched from as further described in thepatent applications WO2005074993 and PCT/EP02/11950. Such modificationcan, in principle, be made to the nucleic acid molecules of the presentinvention at any position thereof. Preferably such modification is madeeither to the 5′-terminal nucleotide, the 3′-terminal nucleotide and/orany nucleotide between the 5′ nucleotide and the 3′ nucleotide of thenucleic acid molecule.

The modification and preferably the PEG and/or HES moiety can beattached to the nucleic acid molecule of the present invention eitherdirectly or through a linker. It is also within the present inventionthat the nucleic acid molecule according to the present inventioncomprises one or more modifications, preferably one or more PEG and/orHES moiety. In an embodiment the individual linker molecule attachesmore than one PEG moiety or HES moiety to a nucleic acid moleculeaccording to the present invention. The linker used in connection withthe present invention can itself be either linear or branched. This kindof linkers are known to the ones skilled in the art and are furtherdescribed in the patent applications WO2005074993 and PCT/EP02/11950.

Without wishing to be bound by any theory, it seems that by modifyingthe nucleic acids according to the present invention with high molecularweight moiety such as a polymer and more particularly the polymersdisclosed herein, which are preferably physiologically acceptable, theexcretion kinetic is changed. More particularly, it seems that due tothe increased molecular weight of such modified inventive nucleic acidsand due to the nucleic acids not being subject to metabolismparticularly when in the L form, excretion from an animal body,preferably from a mammalian body and more preferably from a human bodyis decreased. As excretion typically occurs via the kidneys, the presentinventors assume that the glomerular filtration rate of the thusmodified nucleic acid is significantly reduced compared to the nucleicacids not having this kind of high molecular weight modification whichresults in an increase in the residence time in the body. In connectiontherewith it is particularly noteworthy that, despite such highmolecular weight modification the specificity of the nucleic acidaccording to the present invention is not affected in a detrimentalmanner. Insofar, the nucleic acids according to the present inventionhave surprising characteristics—which normally cannot be expected frompharmaceutically active compounds—such that a pharmaceutical formulationproviding for a sustained release is not necessarily required to providefor a sustained release. Rather the nucleic acids according to thepresent invention in their modified form comprising a high molecularweight moiety, can as such already be used as a sustainedrelease-formulation. Insofar, the modification(s) of the nucleic acidmolecules as disclosed herein and the thus modified nucleic acidmolecules and any composition comprising the same may provide for adistinct, preferably controlled pharmacokinetics and biodistributionthereof. This also includes residence time in circulation anddistribution to tissues. Such modifications are further described in thepatent application PCT/EP02/11950.

However, it is also within the present invention that the nucleic acidsdisclosed herein do not comprise any modification and particularly nohigh molecular weight modification such as PEGylation or HESylation.Such embodiment is particularly preferred when the nucleic acid showspreferential distribution to any target organ or tissue in the body.Nucleic acid agents with such a distributive profile would allowestablishment of effective local concentrations in the target tissuewhile keeping systemic concentration low. This would allow the use oflow doses which is not only beneficial from an economic point of view,but also reduces unnecessary exposure of other tissues to the nucleicacid agent, thus reducing the potential risk of side effects.

The inventive nucleic acids, which are also referred to herein as thenucleic acids according to the present invention, and/or the antagonistsaccording to the present invention may be used for the generation ormanufacture of a medicament. Such medicament or a pharmaceuticalcomposition according to the present invention contains at least one ofthe inventive nucleic acids, optionally together with furtherpharmaceutically active compounds, whereby the inventive nucleic acidpreferably acts as pharmaceutically active compound itself. Suchmedicaments comprise in preferred embodiments at least apharmaceutically acceptable carrier. Such carrier may be, e.g., water,buffer, PBS, glucose solution, preferably a 5% glucose salt balancedsolution, starch, sugar, gelatine or any other acceptable carriersubstance. Such carriers are generally known to the one skilled in theart. It will be acknowledged by the person skilled in the art that anyembodiments, use and aspects of or related to the medicament of thepresent invention is also applicable to the pharmaceutical compositionof the present invention and vice versa.

The indication, diseases and disorders for the treatment and/orprevention of which the nucleic acids, the pharmaceutical compositionsand medicaments in accordance with or prepared in accordance with thepresent invention result from the involvement, either direct orindirect, of MCP-1 in the respective pathogenetic mechanism. However,also those indications, diseases and disorders can be treated andprevented in the pathogenetic mechanism of which MCP-2, MCP-3, MCP-4and/or eotaxin are either directly or indirectly involved. It is obviousfor the ones skilled in the art that particularly those nucleic acidsaccording to the present invention can be used insofar, i.e. for thediseases involving in the broader sense MCP-2, MCP-3, MCP-4 and eotaxin,which interact and bind, respectively, to or with MCP-2, MCP-3, MCP-4and eotaxin, respectively.

More specifically, such uses arise, among others, from the expressionpattern of MCP-1 which suggests that it plays important roles in humandiseases that are characterized by mononuclear cell infiltration. Suchcell infiltration is present in many inflammatory and autoimmunediseases.

In animal models, MCP-1 has been shown to be expressed in the brainafter focal ischemia (Kim 1995; Wang 1995) and during experimentalautoimmune encephalomyelitis (Hulkower 1993; Ransohoff 1993; Banisor2005). MCP-1 may be an important chemokine that targets mononuclearcells in the disease process illustrated by these animal models, such asstroke and multiple sclerosis.

A large body of evidence argues in favor of a unique role of theMCP-1/CCR2 axis in monocyte chemoattraction and thus chronicinflammation: (i) MCP-1- or CCR2-deficient mice show markedly reducedmacrophage chemotactic response while otherwise appearing normal (Kuziel1997; Kurihara 1997; Boring 1997; Lu 1998), (ii), despite functionalredundancy with other chemokines in vitro, loss of MCP-1 effectorfunction alone is sufficient to impair monocytic trafficking in severalinflammatory models (Lloyd 1997; Furuichi 2003; Egashira 2002; Galasso2000; Ogata 1997; Kennedy 1998; Gonzalo 1998; Kitamoto 2003), (iii),MCP-1 levels are elevated in many inflammatory diseases. In fact, MCP-1is thought to play a role in many diseases with and without an obviousinflammatory component such as rheumatoid arthritis (Koch 1992; Hosaka1994; Akahoshi 1993; Harigai 1993; Rollins 1996), renal disease (Wada1996; Viedt 2002), restenosis after angioplasty (Economou 2001), allergyand asthma (Alam 1996; Holgate 1997; Gonzalo 1998), cancer (Salcedo2000; Gordillo 2004), atherosclerosis (Nelken 1991; Yla-Herttuala 1991;Schwartz 1993; Takeya 1993; Boring 1998), psoriasis (Vestergaard 2004),inflammation of the nervous system (Huang 2001), atopic dermatitis(Kaburagi 2001), colitis (Okuno 2002), endometriosis (Jolicoeur 2001),uveitis (Tuaillon 2002), retinal disorders (Nakazawa 2007), idiopathicpulmonary fibrosis and sarcoidosis (Iyonaga 1994) andpolymyositis/dermatomyositis (De Bleecker 2002).

Therapeutic intervention with anti-MCP-1 agents—or CCR2antagonists—would affect the excess inflammatory monocyte traffickingbut may spare basal trafficking of phagocytes, thereby avoiding generalimmunosuppression and increased risk of infections (Dawson 2003).

Additionally, based on the increasing knowledge on the molecularmechanisms of the inflammatory process and the interplay of locallysecreted mediators of inflammation, new targets for the therapy ofkidney diseases have been identified (Holdsworth 2000; Segerer 2000).

One of those targets, for which robust data on expression andinterventional studies with specific antagonists in appropriate animalmodels exist is MCP-1. This protein has a widely non-redundant role forimmune-cell recruitment to sites of renal inflammation. Infiltration ofimmune cells to the kidney is thought to be a major mechanism ofstructural renal damage and decline of renal function in the developmentof various forms of kidney disease.

All types of renal cells can express chemokines including MCP-1 uponstimulation in vitro (Segerer 2000); there is a long list of stimulithat trigger MCP-1 expression in vitro including cytokines, oxygenradicals, immune complexes, and lipid mediators.

In healthy kidneys of rats and mice, MCP-1 is not expressed, but isreadily upregulated during the course of acute and chronic rodent modelsof renal inflammation including immune complex glomerulonephritis, rapidprogressive glomerulonephritis, proliferative glomerulonephritis,diabetic nephropathy, obstructive nephropathy, or acute tubular necrosis(Segerer 2000; Anders 2003). The expression data for MCP-1 in rodents docorrelate well with the respective expression found in human renalbiopsies (Rovin 1994; Cockwell 1998; Wada 1999). Furthermore, renalexpression in human kidneys is associated with disease activity anddeclines when appropriate therapy induced disease remission (Amann2003).

Glomerular mononuclear cell infiltration is associated with thedevelopment of a diffuse glomerulosclerosis in patients with diabeticnephropathy. MCP-1 plays an important role in the recruitment andaccumulation of monocytes and lymphocytes within the glomerulus (Banba2000; Morii 2003).

Locally produced MCP-1 seems to be particularly involved in theinitiation and progression of tubulointerstitial damage, as documentedin experiments using transgenic mice with nephrotoxic serum-inducednephritis (NSN). MCP-1 was mainly detected in vascular endothelialcells, tubular epithelial cells and infiltrated mononuclear cells in theinterstitial lesions. The MCP-1 mediated activation of tubularepithelial cells is consistent with the notion that MCP-1 contributes totubulointerstitial inflammation, a hallmark of progressive renal disease(Wada 2001; Viedt 2002)

Due to the homology between MCP-1 on the one hand and MCP-2, MCP-3,MCP-4 and eotaxin on the other hand, the nucleic acids according to thepresent invention, at least those of them which interact with or bind toMCP-2, MCP-3, MCP-4 and eotaxin, respectively, can typically be used forthe treatment, prevention and/or diagnosis of any disease where MCP-2,MCP-3, MCP-4 and eotaxin, respectively, is either directly or indirectlyinvolved. Involved as preferably used herein, means that if therespective molecule which is involved in the disease, is prevented fromexerting one, several or all of its functions in connection with thepathogenetic mechanism underlying the disease, the disease will be curedor the extent thereof decreased or the outbreak thereof prevented, atleast the symptoms or any indicator of such disease will be relieved andimproved, respectively, such that the symptoms and indicator,respectively, is identical or closer to the one(s) observed in a subjectnot suffering from the disease or not being at risk to develop suchdisease.

Of course, because the MCP-1 binding nucleic acids according to thepresent invention interact with or bind to human or murine MCP-1, askilled person will generally understand that the MCP-1 binding nucleicacids according to the present invention can easily be used for thetreatment, prevention and/or diagnosis of any disease as describedherein of humans and animals.

These members of the monocyte chemoattractant protein (MCP) family, i.e.MCP-2, MCP-3, MCP-4 and eotaxin thus share a high degree of sequencesimilarity with MCP-1. Although not exclusively, eotaxin, MCP-2, -3, and-4 interact via CCR3, the characteristic chemokine receptor on humaneosinophils (Heath 1997). The CCR3 receptor is upregulated in neoplasticconditions, such as cutaneous T-cell lymphoma (Kleinhans 2003),glioblastoma (Kouno 2004), or renal cell carcinoma (Johrer 2005).

More specifically, increased levels of eotaxin are directly associatedwith asthma diagnosis and compromised lung function (Nakamura 1999).Elevated expression of eotaxin at sites of allergic inflammation hasbeen observed in both atopic and nonatopic asthmatics (Ying 1997; Ying1999). Also, mRNAs coding for MCP-2 and -4 are constitutively expressedin a variety of tissues; their physiological functions in thesecontexts, however, are unknown. Plasma MCP-2 levels are elevated insepsis together with MCP-1 (Bossink 1995); MCP-3 expression occurs inasthmatics (Humbert 1997). Finally. MCP-4 can be found at the luminalsurface of atherosclerotic vessels (Berkhout 1997).

Accordingly, disease and/or disorders and/or diseased conditions for thetreatment and/or prevention of which the medicament according to thepresent invention may be used include, but are not limited toinflammatory diseases, autoimmune diseases, autoimmuneencephalomyelitis, stroke, acute and chronic multiple sclerosis, chronicinflammation, rheumatoid arthritis, renal diseases, restenosis,restenosis after angioplasty, acute and chronic allergic reactions,primary and secondary immunologic or allergic reactions, asthma,conjunctivitis, bronchitis, cancer, atherosclerosis, artherioscleroticcardiovasular heart failure or stroke, psoriasis, psoriatic arthritis,inflammation of the nervous system, atopic dermatitis, colitis,endometriosis, uveitis, retinal disorders including maculardegeneration, retinal detachment, diabetic retinopathy, retinopathy ofprematurity, retinitis pigmentosa, proliferative vitreoretinopathy, andcentral serous chorioretinopathy; idiopathic pulmonary fibrosis,sarcoidosis, polymyositis, dermatomyositis, avoidance ofimmunosuppression, reducing the risk of infection, sepsis, renalinflammation, glomerulonephritis, rapid progressive glomerulonephritis,proliferative glomerulonephritis, diabetic nephropathy, obstructivenephropathy, acute tubular necrosis, and diffuse glomerulosclerosis,systemic lupus erythematosus, chronic bronchitis, Behçet's disease,amyotrophic lateral sclerosis (ALS), premature atherosclerosis afterKawasaki's disease, myocardial infarction, obesity, chronic liverdisease, peyronie's disease, acute spinal chord injury, lung or kidneytransplantation, myocarditis, Alzheimer's disease, and neuropathy,breast carcinoma, gastric carcinoma, bladder cancer, ovarian cancer,hamartoma, colorectal carcinoma, colonic adenoma, pancreatitis, chronicobstructive pulmonary disease (COPD) and inflammatory bowel diseasessuch as Crohn's disease or ulcerative colitis.

In a further embodiment, the medicament comprises a furtherpharmaceutically active agent. Such further pharmaceutically activecompounds are, among others but not limited thereto, those known tocontrol blood pressure and diabetes such as angiotensin convertingenzyme (ACE) inhibitors and angiotensin receptor blockers. The furtherpharmaceutically active compound can be, in a further embodiment, alsoone of those compounds which reduce infiltration of immune cells tosites of chronic inflammation or generally suppress the exuberant immuneresponse that is present in chronic inflammatory settings and that leadsto tissue damage. Such compounds can be, but are not limited to,steroids or immune suppressants and are preferably selected from thegroup comprising corticosteroids like prednisone, methylprednisolone,hydrocortisone, dexamethasone and general immunosuppressants such ascyclophosphamide, cyclosporine, chlorambucil, azathioprine, tacrolimusor mycophenolate mofetil. Additionally, more specific blockers of T-cellcostimulation, e.g. blockers of CD154 or CD40 or CD28 or CD86 or CD80;or T- and/or B-cell depleting agents like an anti-CD20 agent are usefulin further embodiments. Finally, the further pharmaceutically activeagent may be a modulator of the activity of any other chemokine whichcan be a chemokine agonist or antagonist or a chemokine receptor agonistor antagonist. Alternatively, or additionally, such furtherpharmaceutically active agent is a further nucleic acid according to thepresent invention. Alternatively, the medicament comprises at least onemore nucleic acid which binds to a target molecule different from MCP-1or exhibits a function which is different from the one of the nucleicacids according to the present invention.

It is within the present invention that the medicament is alternativelyor additionally used, in principle, for the prevention of any of thediseases disclosed in connection with the use of the medicament for thetreatment of said diseases. Respective markers therefore, i.e. for therespective diseases are known to the ones skilled in the art.Preferably, the respective marker is MCP-1. Alternatively and/oradditionally, the respective marker is selected from the groupcomprising MCP-2, MCP-3, MCP-4 and eotaxin. A still further group ofmarkers is selected from the group comprising autoreactive antibodies inthe plasma, such as, for example, anti-dsDNA antibodies or rheumatoidfactor.

In one embodiment of the medicament of the present invention, suchmedicament is for use in combination with other treatments for any ofthe diseases disclosed herein, particularly those for which themedicament of the present invention is to be used.

“Combination therapy” (or “co-therapy”) includes the administration of amedicament of the invention and at least a second agent as part of aspecific treatment regimen intended to provide the beneficial effectfrom the co-action of these therapeutic agents, i. e. the medicament ofthe present invention and said second agent. The beneficial effect ofthe combination includes, but is not limited to, pharmacokinetic orpharmacodynamic co-action resulting from the combination of therapeuticagents. Administration of these therapeutic agents in combinationtypically is carried out over a defined time period (usually minutes,hours, days or weeks depending upon the combination selected).

“Combination therapy” may, but generally is not, intended to encompassthe administration of two or more of these therapeutic agents as part ofseparate monotherapy regimens that incidentally and arbitrarily resultin the combinations of the present invention. “Combination therapy” isintended to embrace administration of these therapeutic agents in asequential manner, that is, wherein each therapeutic agent isadministered at a different time, as well as administration of thesetherapeutic agents, or at least two of the therapeutic agents, in asubstantially simultaneous manner. Substantially simultaneousadministration can be accomplished, for example, by administering to asubject a single capsule having a fixed ratio of each therapeutic agentor in multiple, single capsules for each of the therapeutic agents.

Sequential or substantially simultaneous administration of eachtherapeutic agent can be effected by any appropriate route including,but not limited to, topical routes, oral routes, intravenous routes,intramuscular routes, and direct absorption through mucous membranetissues. The therapeutic agents can be administered by the same route orby different routes. For example, a first therapeutic agent of thecombination selected may be administered by injection while the othertherapeutic agents of the combination may be administered topically.

Alternatively, for example, all therapeutic agents may be administeredtopically or all therapeutic agents may be administered by injection.The sequence in which the therapeutic agents are administered is notnarrowly critical unless noted otherwise. “Combination therapy” also canembrace the administration of the therapeutic agents as described abovein further combination with other biologically active ingredients. Wherethe combination therapy further comprises a non-drug treatment, thenon-drug treatment may be conducted at any suitable time so long as abeneficial effect from the co-action of the combination of thetherapeutic agents and non-drug treatment is achieved. For example, inappropriate cases, the beneficial effect is still achieved when thenon-drug treatment is temporally removed from the administration of thetherapeutic agents, perhaps by days or even weeks.

As outlined in general terms above, the medicament according to thepresent invention can be administered, in principle, in any form knownto the ones skilled in the art. A preferred route of administration issystemic administration, more preferably by parenteral administration,preferably by injection. Alternatively, the medicament may beadministered locally. Other routes of administration compriseintramuscular, intraperitoneal, and subcutaneous, per orum, intranasal,intratracheal or pulmonary with preference given to the route ofadministration that is the least invasive, while ensuring efficiency.

Parenteral administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Additionally, oneapproach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, that are well known to theordinary skill in the art.

Furthermore, preferred medicaments of the present invention can beadministered in intranasal form via topical use of suitable intranasalvehicles, inhalants, or via transdermal routes, using those forms oftransdermal skin patches well known to those of ordinary skill in thatart. To be administered in the form of a transdermal delivery system,the dosage administration will, of course, be continuous rather thanintermittent throughout the dosage regimen. Other preferred topicalpreparations include creams, ointments, lotions, aerosol sprays andgels, wherein the concentration of active ingredient would typicallyrange from 0.01% to 15%, w/w or w/v.

The medicament of the present invention will generally comprise aneffective amount of the active component(s) of the therapy, including,but not limited to, a nucleic acid molecule of the present invention,dissolved or dispersed in a pharmaceutically acceptable medium.Pharmaceutically acceptable media or carriers include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Supplementary active ingredients can also be incorporatedinto the medicament of the present invention.

In a further aspect the present invention is related to a pharmaceuticalcomposition. Such pharmaceutical composition comprises at least one ofthe nucleic acids according to the present invention and preferably apharmaceutically acceptable vehicle. Such vehicle can be any vehicle orany binder used and/or known in the art. More particularly such binderor vehicle is any binder or vehicle as discussed in connection with themanufacture of the medicament disclosed herein. In a further embodiment,the pharmaceutical composition comprises a further pharmaceuticallyactive agent.

The preparation of a medicament and a pharmaceutical composition will beknown to those of skill in the art in light of the present disclosure.Typically, such compositions may be prepared as injectables, either usliquid solutions or suspensions; solid forms suitable for solution in,or suspension in, liquid prior to injection; as tablets or other solidsfor oral administration; as time release capsules; or in any other formcurrently used, including eye drops, creams, lotions, salves, inhalantsand the like. The use of sterile formulations, such as saline-basedwashes, by surgeons, physicians or health care workers to treat aparticular area in the operating field may also be particularly useful.Compositions may also be delivered via microdevice, microparticle orsponge.

Upon formulation, a medicament will be administered in a mannercompatible with the dosage formulation, and in such amount as ispharmacologically effective. The formulations are easily administered ina variety of dosage forms, such as the type of injectable solutionsdescribed above, but drug release capsules and the like can also beemployed.

In this context, the quantity of active ingredient and volume ofcomposition to be administered depends on the individual or the subjectto be treated. Specific amounts of active compound required foradministration depend on the judgment of the practitioner and arepeculiar to each individual.

A minimal volume of a medicament required to disperse the activecompounds is typically utilized. Suitable regimes for administration arealso variable, but would be typified by initially administering thecompound and monitoring the results and then giving further controlleddoses at further intervals.

For instance, for oral administration in the form of a tablet or capsule(e.g., a gelatin capsule), the active drug component, i. e. a nucleicacid molecule of the present invention and/or any furtherpharmaceutically active agent, also referred to herein as therapeuticagent(s) or active compound(s) can be combined with an oral, non-toxic,pharmaceutically acceptable inert carrier such as ethanol, glycerol,water and the like. Moreover, when desired or necessary, suitablebinders, lubricants, disintegrating agents, and coloring agents can alsobe incorporated into the mixture. Suitable binders include starch,magnesium aluminum silicate, starch paste, gelatin, methylcellulose,sodium carboxymethylcellulose and/or polyvinylpyrrolidone, naturalsugars such as glucose or beta-lactose, corn sweeteners, natural andsynthetic gums such as acacia, tragacanth or sodium alginate,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, silica, talcum,stearic acid, its magnesium or calcium salt and/or polyethyleneglycol,and the like. Disintegrators include, without limitation, starch, methylcellulose, agar, bentonite, xanthan gum starches, agar, alginic acid orits sodium salt, or effervescent mixtures, and the like. Diluents,include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, celluloseand/or glycine.

The medicament of the invention can also be administered in such oraldosage forms as timed release and sustained release tablets or capsules,pills, powders, granules, elixirs, tinctures, suspensions, syrups andemulsions. Suppositories are advantageously prepared from fattyemulsions or suspensions.

The pharmaceutical composition or medicament may be sterilized and/orcontain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating, or coating methods, andtypically contain about 0.1% to 75%, preferably about 1% to 50%, of theactive ingredient.

Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc. The active compound isdissolved in or mixed with a pharmaceutically pure solvent such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, to thereby form the injectable solution or suspension.Additionally, solid forms suitable for dissolving in liquid prior toinjection can be formulated.

For solid compositions, excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. Theactive compound defined above, may be also formulated as suppositories,using for example, polyalkylene glycols, for example, propylene glycol,as the carrier. In some embodiments, suppositories are advantageouslyprepared from fatty emulsions or suspensions.

The medicaments and nucleic acid molecules, respectively, of the presentinvention can also be administered in the form of liposome deliverysystems, such as small unilamellar vesicles, large unilamellar vesiclesand multilamellar vesicles. Liposomes can be formed from a variety ofphospholipids, containing cholesterol, stearylamine orphosphatidylcholines. In some embodiments, a film of lipid components ishydrated with an aqueous solution of drug to a form lipid layerencapsulating the drug, what is well known to the ordinary skill in theart. For example, the nucleic acid molecules described herein can beprovided as a complex with a lipophilic compound or non-immunogenic,high molecular weight compound constructed using methods known in theart. Additionally, liposomes may bear such nucleic acid molecules ontheir surface for targeting and carrying cytotoxic agents internally tomediate cell killing. An example of nucleic-acid associated complexes isprovided in U.S. Pat. No. 6,011,020.

The medicaments and nucleic acid molecules, respectively, of the presentinvention may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinylpyrrolidone, pyrancopolymer, polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the medicaments andnucleic acid molecules, respectively, of the present invention may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drag, for example, polylactic acid, polyepsiloncapro lactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

If desired, the pharmaceutical composition and medicament, respectively,to be administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and other substances such as for example, sodium acetate, andtriethanolamine oleate.

The dosage regimen utilizing the nucleic acid molecules and medicaments,respectively, of the present invention is selected in accordance with avariety of factors including type, species, age, weight, sex and medicalcondition of the patient; the severity of the condition to be treated;the route of administration the renal and hepatic function of thepatient; and the particular aptamer or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Effective plasma levels of the nucleic acid according to the presentinvention preferably range from 500 fM to 500 μM in the treatment of anyof the diseases disclosed herein.

The nucleic acid molecules and medicaments, respectively, of the presentinvention may preferably be administered in a single daily dose, everysecond or third day, weekly, every second week, in a single monthly doseor every third month.

It is within the present invention that the medicament as describedherein constitutes the pharmaceutical composition disclosed herein.

In a further aspect the present invention is related to a method for thetreatment of a subject who is need of such treatment, whereby the methodcomprises the administration of a pharmaceutically active amount of atleast one of the nucleic acids according to the present invention. In anembodiment, the subject suffers from a disease or is at risk to developsuch disease, whereby the disease is any of those disclosed herein,particularly any of those diseases disclosed in connection with the useof any of the nucleic acids according to the present invention for themanufacture of a medicament.

It is to be understood that the nucleic acid as well as the antagonistsaccording to the present invention can be used not only as a medicamentor for the manufacture of a medicament, but also for cosmetic purposes,particularly with regard to the involvement of MCP-1 in inflamedregional skin lesions. Therefore, a further condition or disease for thetreatment or prevention of which the nucleic acid, the medicament and/orthe pharmaceutical composition according to the present invention can beused, is inflamed regional skin lesions.

As preferably used herein a diagnostic or diagnostic agent or diagnosticmeans is suitable to detect, either directly or indirectly MCP-1,preferably MCP-1 as described herein and more preferably MCP-1 asdescribed herein in connection with the various disorders and diseasesdescribed herein. However, to the extent that the nucleic acid moleculesaccording to the present invention are also binding to any, some or allof MCP-2, MCP-3, MCP-4 and eotaxin, such nucleic acid molecules can alsobe used for the diagnosis of diseases and disorders, respectively, thepathogenetic mechanism is either directly or indirectly linked orassociated with the over-expression or over-activity with MCP-2, MCP-3,MCP-4 and/or eotaxin. The diagnostic is suitable for the detectionand/or follow-up of any of the disorders and diseases, respectively,described herein. Such detection is possible through the binding of thenucleic acids according to the present invention to MCP-1. Such bindingcan be either directly or indirectly be detected.

The respective methods and means are known to the ones skilled in theart. Among others, the nucleic acids according to the present inventionmay comprise a label which allows the detection of the nucleic acidsaccording to the present invention, preferably the nucleic acid bound toMCP-1. Such a label is preferably selected from the group comprisingradioactive, enzymatic and fluorescent labels. In principle, all knownassays developed for antibodies can be adopted for the nucleic acidsaccording to the present invention whereas the target-binding antibodyis substituted to a target-binding nucleic acid. In antibody-assaysusing unlabeled target-binding antibodies the detection is preferablydone by a secondary antibody which is modified with radioactive,enzymatic and fluorescent labels and bind to the target-binding antibodyat its Fc-fragment. In the case of a nucleic acid, preferably a nucleicacid according to the present invention, the nucleic acid is modifiedwith such a label, whereby preferably such a label is selected from thegroup comprising biotin, Cy-3 and Cy-5, and such label is detected by anantibody directed against such label, e.g. an anti-biotin antibody, ananti-Cy3 antibody or an anti-Cy5 antibody, or—in the case that the labelis biotin—the label is detected by streptavidin or avidin whichnaturally bind to biotin. Such antibody, streptavidin or avidin in turnis preferably modified with a respective label, e.g. a radioactive,enzymatic or fluorescent label (like an secondary antibody).

In a further embodiment the nucleic acid molecules according to theinvention are detected or analysed by a second detection means, whereinthe said detection means is a molecular beacon. The methodology ofmolecular beacon is known to persons skilled in the art. In brief,nucleic acids probes which are also referred to as molecular beacons,are a reverse complement to the nucleic acids sample to be detected andhybridise because of this to a part of the nucleic acid sample to bedetected. Upon binding to the nucleic acid sample the fluorophoricgroups of the molecular beacon are separated which results in a changeof the fluorescence signal, preferably a change in intensity. Thischange correlates with the amount of nucleic acids sample present.

It will be acknowledged that the detection of MCP-1 using the nucleicacids according to the present invention will particularly allow thedetection of MCP-1 as defined herein.

In connection with the detection of the MCP-1 a preferred methodcomprises the following steps:

-   -   (a) providing a sample which is to be tested for the presence of        MCP-1,    -   (b) providing a nucleic acid according to the present invention,    -   (c) reacting the sample with the nucleic acid, preferably in a        reaction vessel    -   whereby step (a) can be performed prior to step (b), or step (b)        can be preformed prior to step (a).

In a preferred embodiment a further step d) is provided, which consistsin the detection of the reaction of the sample with the nucleic acid.Preferably, the nucleic acid of step b) is immobilised to a surface. Thesurface may be the surface of a reaction vessel such as a reaction tube,a well of a plate, or the surface of a device contained in such reactionvessel such as, for example, a bead. The immobilisation of the nucleicacid to the surface can be made by any means known to the ones skilledin the art including, but not limited to, non-covalent or covalentlinkages. Preferably, the linkage is established via a covalent chemicalbond between the surface and the nucleic acid. However, it is alsowithin the present invention that the nucleic acid is indirectlyimmobilised to a surface, whereby such indirect immobilisation involvesthe use of a further component or a pair of interaction partners. Suchfurther component is preferably a compound which specifically interactswith the nucleic acid to be immobilised which is also referred to asinteraction partner, and thus mediates the attachment of the nucleicacid to the surface. The interaction partner is preferably selected fromthe group comprising nucleic acids, polypeptides, proteins andantibodies. Preferably, the interaction partner is an antibody, morepreferably a monoclonal antibody. Alternatively, the interaction partneris a nucleic acid, preferably a functional nucleic acid. More preferablysuch functional nucleic acid is selected from the group comprisingaptamers, spiegelmers, and nucleic acids which are at least partiallycomplementary to the nucleic acid. In a further alternative embodiment,the binding of the nucleic acid to the surface is mediated by amulti-partite interaction partner. Such multi-partite interactionpartner is preferably a pair of interaction partners or an interactionpartner consisting of a first member and a second member, whereby thefirst member is comprised by or attached to the nucleic acid and thesecond member is attached to or comprised by the surface. Themulti-partite interaction partner is preferably selected from the groupof pairs of interaction partners comprising biotin and avidin, biotinand streptavidin, and biotin and neutravidin. Preferably, the firstmember of the pair of interaction partners is biotin.

A preferred result of such method is the formation of an immobilisedcomplex of MCP-1 and the nucleic acid, whereby more preferably saidcomplex is detected. It is within an embodiment that from the complexthe MCP-1 is detected.

A respective detection means which is in compliance with thisrequirement is, for example, any detection means which is specific forthat/those part(s) of the MCP-1. A particularly preferred detectionmeans is a detection means which is selected from the group comprisingnucleic acids, polypeptides, proteins and antibodies, the generation ofwhich is known to the ones skilled in the art.

The method for the detection of MCP-1 also comprises that the sample isremoved from the reaction vessel which has preferably been used toperform step c).

The method comprises in a further embodiment also the step ofimmobilising an interaction partner of MCP-1 on a surface, preferably asurface as defined above, whereby the interaction partner is defined asherein and preferably as above in connection with the respective methodand more preferably comprises nucleic acids, polypeptides, proteins andantibodies in their various embodiments. In this embodiment, aparticularly preferred detection means is a nucleic acid according tothe present invention, whereby such nucleic acid may preferably belabelled or non-labelled. In case such nucleic acid is labelled it candirectly or indirectly be detected. Such detection may also involve theuse of a second detection means which is, preferably, also selected fromthe group comprising nucleic acids, polypeptides, proteins andembodiments in the various embodiments described herein. Such detectionmeans are preferably specific for the nucleic acid according to thepresent invention. In a more preferred embodiment, the second detectionmeans is a molecular beacon. Either the nucleic acid or the seconddetection means or both may comprise in a preferred embodiment adetection label. The detection label is preferably selected from thegroup comprising biotin, a bromo-desoxyuridine label, a digoxigeninlabel, a fluorescence label, a UV-label, a radio-label, and a chelatormolecule. Alternatively, the second detection means interacts with thedetection label which is preferably contained by, comprised by orattached to the nucleic acid. Particularly preferred combinations are asfollows:

-   -   the detection label is biotin and the second detection means is        an antibody directed against biotin, or wherein    -   the detection label is biotin and the second detection means is        an avidin or an avidin carrying molecule, or wherein    -   the detection label is biotin and the second detection means is        a streptavidin or a stretavidin carrying molecule, or wherein    -   the detection label is biotin and the second detection means is        a neutravidin or a neutravidin carrying molecule, or    -   wherein the detection label is a bromo-desoxyuridine and the        second detection means is an antibody directed against        bromo-desoxyuridine, or wherein    -   the detection label is a digoxigenin and the second detection        means is an antibody directed against digoxigenin, or wherein    -   the detection label is a chelator and the second detection means        is a radio-nuclide, whereby it is preferred that said detection        label is attached to the nucleic acid. It is to be acknowledged        that this kind of combination is also applicable to the        embodiment where the nucleic acid is attached to the surface. In        such embodiment it is preferred that the detection label is        attached to the interaction partner.

Finally, it is also within the present invention that the seconddetection means is detected using a third detection means, preferablythe third detection means is an enzyme, more preferably showing anenzymatic reaction upon detection of the second detection means, or thethird detection means is a means for detecting radiation, morepreferably radiation emitted by a radio-nuclide. Preferably, the thirddetection means is specifically detecting and/or interacting with thesecond detection means.

Also in the embodiment with an interaction partner of MCP-1 beingimmobilised on a surface and the nucleic acid according to the presentinvention is preferably added to the complex formed between theinteraction partner and the MCP-1, the sample can be removed from thereaction, more preferably from the reaction vessel where step c) and/ord) are preformed.

In an embodiment the nucleic acid according to the present inventioncomprises a fluorescence moiety and whereby the fluorescence of thefluorescence moiety is different upon complex formation between thenucleic acid and MCP-1 and free MCP-1.

In a further embodiment the nucleic acid is a derivative of the nucleicacid according to the present invention, whereby the derivative of thenucleic acid comprises at least one fluorescent derivative of adenosinereplacing adenosine. In a preferred embodiment the fluorescentderivative of adenosine is ethenoadenosine.

In a further embodiment the complex consisting of the derivative of thenucleic acid according to the present invention and the MCP-1 isdetected using fluorescence.

In an embodiment of the method a signal is created in step (c) or step(d) and preferably the signal is correlated with the concentration ofMCP-1 in the sample.

In a preferred aspect, the assays may be performed in 96-well plates,where components are immobilized in the reaction vessels as describedabove and the wells acting as reaction vessels.

It will be acknowledged by the ones skilled in the art that what hasbeen said above also applies to MCP-2, MCP-3, MCP-4 and/or eotaxin, atleast to the extent that the nucleic acids according to the presentinvention are also binding to or with MCP-2, MCP-3, MCP-4 and/oreotaxin.

The inventive nucleic acid may further be used as starting material fordrug design. Basically there are two possible approaches. One approachis the screening of compound libraries whereas such compound librariesare preferably low molecular weight compound libraries. In anembodiment, the screening is a high throughput screening. Preferably,high throughput screening is the fast, efficient, trial-and-errorevaluation of compounds in a target based assay. In best case theanalysis are carried by a colorimetric measurement. Libraries as used inconnection therewith are known to the one skilled in the art.

Alternatively, the nucleic acid according to the present invention maybe used for rational design of drugs. Preferably, rational drug designis the design of a pharmaceutical lead structure. Starting from the3-dimensional structure of the target which is typically identified bymethods such as X-ray crystallography or nuclear magnetic resonancespectroscopy, computer programs are used to search through databasescontaining structures of many different chemical compounds. Theselection is done by a computer, the identified compounds cansubsequently be tested in the laboratory.

The rational design of drugs may start from any of the nucleic acidaccording to the present invention and involves a structure, preferablya three dimensional structure, which is similar to the structure of theinventive nucleic acids or identical to the binding mediating parts ofthe structure of the inventive nucleic acids. In any case such structurestill shows the same or a similar binding characteristic as theinventive nucleic acids. In either a further step or as an alternativestep in the rational design of drugs the preferably three dimensionalstructure of those parts of the nucleic acids binding to theneurotransmitter are mimicked by chemical groups which are differentfrom nucleotides and nucleic acids. By this mimicry a compound differentfrom the nucleic acids can be designed. Such compound is preferably asmall molecule or a peptide.

In case of screening of compound libraries, such as by using acompetitive assay which are known to the one skilled in the arts,appropriate MCP-1 analogues, MCP-1 agonists or MCP-1 antagonists may befound. Such competitive assays may be set up as follows. The inventivenucleic acid, preferably a spiegelmer which is a target bindingL-nucleic acid, is coupled to a solid phase. In order to identify MCP-1analogues labelled MCP-1 may be added to the assay. A potential analoguewould compete with the MCP-1 molecules binding to the spiegelmer whichwould go along with a decrease in the signal obtained by the respectivelabel. Screening for agonists or antagonists may involve the use of acell culture assay as known to the ones skilled in the art.

The kit according to the present invention may comprise at least one orseveral of the inventive nucleic acids. Additionally, the kit maycomprise at least one or several positive or negative controls. Apositive control may, for example, be MCP-1, particularly the oneagainst which the inventive nucleic acid is selected or to which itbinds, preferably, in liquid form. A negative control may, e.g., be apeptide which is defined in terms of biophysical properties similar toMCP-1, but which is not recognized by the inventive nucleic acids.Furthermore, said kit may comprise one or several buffers. The variousingredients may be contained in the kit in dried or lyophilised form orsolved in a liquid. The kit may comprise one or several containers whichin turn may contain one or several ingredients of the kit. In a furtherembodiment, the kit comprises an instruction or instruction leafletwhich provides to the user information on how to use the kit and itsvarious ingredients.

The pharmaceutical and bioanalytical determination of the nucleic acidaccording to the present invention is elementarily for the assessment ofits pharmacokinetic and biodynamic profile in several humours, tissuesand organs of the human and non-human body. For such purpose, any of thedetection methods disclosed herein or known to a person skilled in thean may be used. In a further aspect of the present invention a sandwichhybridisation assay for the detection of the nucleic acid according tothe present invention is provided. Within the detection assay a captureprobe and a detection probe are used. The capture probe is complementaryto the first part and the detection probe to the second part of thenucleic acid according to the present invention. Both, capture anddetection probe, can be formed by DNA nucleotides, modified DNAnucleotides, modified RNA nucleotides, RNA nucleotides, LNA nucleotidesand/or PNA nucleotides.

Hence, the capture probe comprise a sequence stretch complementary tothe 5′-end of the nucleic acid according to the present invention andthe detection probe comprise a sequence stretch complementary to the3′-end of the nucleic acid according to the present invention. In thiscase the capture probe is immobilised to a surface or matrix via its5′-end whereby the capture probe can be immobilised directly at its5′-end or via a linker between of its 5′-end and the surface or matrix.However, in principle the linker can be linked to each nucleotide of thecapture probe. The linker can be formed by hydrophilic linkers ofskilled in the art or by D-DNA nucleotides, modified D-DNA nucleotides,D-RNA nucleotides, modified D-RNA nucleotides, D-LNA nucleotides. PNAnucleotides, L-RNA nucleotides, L-DNA nucleotides, modified L-RNAnucleotides, modified L-DNA nucleotides and/or L-LNA nucleotides.

Alternatively, the capture probe comprises a sequence stretchcomplementary to the 3′-end of the nucleic acid according to the presentinvention and the detection probe comprise a sequence stretchcomplementary to the 5′-end of the nucleic acid according to the presentinvention. In this case the capture probe is immobilised to a surface ormatrix via its 3′-end whereby the capture probe can be immobiliseddirectly at its 3′-end or via a linker between of its 3′-end and thesurface or matrix. However, in principle, the linker can be linked toeach nucleotide of the sequence stretch that is complementary to thenucleic acid according to the present invention. The linker can beformed by hydrophilic linkers of skilled in the art or by D-DNAnucleotides, modified D-DNA nucleotides, D-RNA nucleotides, modifiedD-RNA nucleotides, D-LNA nucleotides, PNA nucleotides, L-RNAnucleotides, L-DNA nucleotides, modified L-RNA nucleotides, modifiedL-DNA nucleotides and/or L-LNA nucleotides.

The number of nucleotides of the capture and detection probe that mayhybridize to the nucleic acid according to the present invention isvariable and can be dependant from the number of nucleotides of thecapture and/or the detection probe and/or the nucleic acid according tothe present invention itself. The total number of nucleotides of thecapture and the detection probe that may hybridise to the nucleic acidaccording to the present invention should be maximal the number ofnucleotides that are comprised by the nucleic acid according to thepresent invention. The minimal number of nucleotides (2 to 10nucleotides) of the detection and capture probe should allowhybridisation to the 5′-end or 3′-end, respectively, of the nucleic acidaccording to the present invention. In order to realize high specificityand selectivity between the nucleic acid according to the presentinvention and other nucleic acids occurring in samples that are analyzedthe total number of nucleotides of the capture and detection probeshould be or maximal the number of nucleotides that are comprised by thenucleic acid according to the present invention.

Moreover the detection probe preferably carries a marker molecule orlabel that can be detected as previously described herein. The label ormarker molecule can in principle be linked to each nucleotide of thedetection probe. Preferably, the label or marker is located at the5′-end or 3′-end of the detection probe, whereby between the nucleotideswithin the detection probe that are complementary to the nucleic acidaccording to the present invention, and the label a linker can beinserted. The linker can be formed by hydrophilic linkers of skilled inthe art or by D-DNA nucleotides, modified D-DNA nucleotides, D-RNAnucleotides, modified D-RNA nucleotides, D-LNA nucleotides, PNAnucleotides, L-RNA nucleotides, L-DNA nucleotides, modified L-RNAnucleotides, modified L-DNA nucleotides and/or L-LNA nucleotides.

The detection of the nucleic acid according to the present invention canbe carried out as follows: The nucleic acid according to the presentinvention hybridises with one of its ends to the capture probe and withthe other end to the detection probe. Afterwards unbound detection probeis removed by, e. g., one or several washing steps. The amount of bounddetection probe which preferably carries a label or marker molecule, canbe measured subsequently.

As preferably used herein, the term treatment comprises in a preferredembodiment additionally or alternatively prevention and/or follow-up.

As preferably used herein, the terms disease and disorder shall be usedin an interchangeable manner, if not indicated to the contrary.

As used herein, the term comprise is preferably not intended to limitthe subject matter followed or described by such term. However, in analternative embodiment the term comprises shall be understood in themeaning of containing and thus as limiting the subject matter followedor described by such term.

The various SEQ. ID. Nos., the chemical nature of the nucleic acidmolecules according to the present invention and the target moleculesMCP-1 as used herein, the actual sequence thereof and the internalreference number is summarized in the following table.

Seq.- RNA/ ID Peptide Sequence Internal Reference 1 L-proteinQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVhuman MCP-1, huMCP-1, QDSMDHLDKQTQTPKT CCL2 2 L-proteinQPDAVNAPLTCCYSFTSKMIPMSRLESYKRITSSRCPKEAVVFVTKLKREVCADPKKEWVmouse MCP-1, mCCL2,QTYIKNLDRNQMRSEPTTLFKTASALRSSAPLNVKLTRKSEANASTTFSTTTSSTSVGVTmMCP-1, murine MCP-1 SVTVN (Mus musculus) 3 L-proteinQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVmonkey MCP-1 (Macaca QDSMDHLDKQIQTPKP mulatta) 4 L-proteinQPDAINSPVTCCYTLTSKKISMQRLMSYRRVTSSKCPKEAVIFKTIAGKEICAEPKQKWV pig MCP-1QDSISHLDKKNQTPKP (Sus scrofa) 5 L-proteinQPDAIISPVTCCYTLTNKKISIQRLASYKRVTSSKCPKEAVIFKTVLNKEICADPKQKWVdog MCP-1 (Canis QDSMAHLDKKSQTQTA familiaris) 6 L-proteinQPDAVNSPVTCCYTFTNKTISVKRLMSYRRINSTKCPKEAVIFMTKLAKGICADPKQKWVrabbit MCP-1 QDAIANLDKKMQTPKTLTSYSTTQEHTTNLSSTRTPSTTTSL (Oryctolaguscuniculus) 7 L-proteinQPVGINTSTTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVhuman MCP-3, CCL7, QDFMKHLDKKTQTPKL huMCP-3 8 L-proteinGPASVPTTCCFNLANRKIPLQRLESYRRITSGKCPQKAVIFKTKLAKDICADPKKKWVQDhuman eotaxin/CCL11 SMKYLDQKSPTPKP 9 L-proteinQPDSVSIPITCCFNVINRKIPIQRLESYTRITNIQCPKEAVIFKTKRGKEVCADPKERWVhuman MCP-2, CCL8, RDSMKHLDQIFQNLKP huMCP-2 10 L-RNAAGCGUGCCCGGAGUGGCAGGGGGACGCGACCUGCAAUAAUGCACGCU 169-B1trc 11 L-RNAAGCGUGCCCGGAGUGGCAGGGGGACGCGACCUGCAAUUGCACGCU 169-F3trc 12 L-RNAAGCGUGCCCGGAGUGGCAGGGGGACGCGACCUGUAAUAAUGCACGCU 169-C1trc 13 L-RNAAGCGUGCCCGGUGUGGCAGGGGGACGCGACCUGCAAUAAUGCGCGCU 169-A3trc 14 L-RNAAGCGUGCCCGGAGUAGCAGGGGGGCGCGACCUGCAAUAAUGCACGCU 169-B2trc 15 L-RNAAGCGUGCCCGGUGUGGUAGGGGGGCGCGAUCUACAAUUGCACGCU 176-B12trc 16 L-RNAAGCGUGCCCGGUGUGACAGGGGGGCGCGACCUGCAUUUGCACGCU 176-D9trc 17 L-RNAAGCGUGCCCGGUGUGGCAGGGGGGCGCGACCUGUAUUUGCACGCU 176-B10trc 18 L-RNAAGCGUGCCCGGAGUGGCAGGGGGGCGCGACCUGCAAUAAUGCACGCU 169-F2trc 19 L-RNAAGCGUGCCCGGUGUGGCAGGGGGGCGCGACCUGCAAUUGCACGCU 176-B9trc 20 L-RNAAGCAUGCCCGGUGUGGCAGGGGGGCGCGACCUGCAUUUGCAUGCU 176-H9trc 21 L-RNAAGCGUGCCCGGUGUGGUAGGGGGGCGCGACCUACAUUUGCACGCU 176-E10trc 22 L-RNAAGUGUGCCAGCUGUGAUGGGGGGGCGCGACCCAUUUUACACACU 176-G9trc 23 L-RNAAGUGUGCCAGCGUGAUGGGGGGGCGCGACCCAUUUUACACACU 176-F9trc 24 L-RNAAGUGUGCGAGCGUGAUGGGGGGGCGCGACCCAUUUUACAUACU 176-C11trc 25 L-RNAAGUGUGCCAGCGUGAUGGGGGGGCGCGACCCAUUUUACAUACU 176-E11trc 26 L-RNAAGUAUGCCAGCGUGAUGGGGGGGCGCGACCCAUUUACAUACU 176-D10trc 27 L-RNAAGUGUGCCAGUGUGAUGGGGGGGCGCGACCCAUUUUACACACU 176-H10trc 28 L-RNAAGCGUGCCAGUGUGAUGGGGGGGCGCGACCCAUUUUACACGCU 176-C9trc 29 L-RNAACGCACGUCCCUCACCGGUGCAAGUGAAGCCGCGGCUCUGCGU 180-B1-001 30 L-RNAACGCACCUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGC 180-A4-002 31 L-RNAACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGU 180-D1-002 32 L-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGU 180-D1-011 33 L-RNAACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGC 180-D1-012 34 L-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGC 180-D1-018 35 L-RNACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGU 180-D1-034 36 L-RNACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG 180-D1-035 37 L-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG 180-D1-036 = NOX-E36 38 L-RNAGUGCUGCGUAGUGGAAGACUACCUAAUGACAGCCGAAUGCUGGCAGCAC 178-A8 39 L-RNAGUGCUGCGUAGUGGAAGACUACCUAAUGACAGCCUAAUGCUGGCAGCAC 178-F7 40 L-RNAGUGCUGCGUAGUGGAAGACUACCUUAUGACAGCCGAAUGCUGGCAGCAC 178-G7 41 L-RNAGUGCUGCGUAGUGAAAAACUACUGCCAGUGGGUCAGAGCUAGCAGCAC 178-C6 42 L-RNAGUGCUGCGGAGUUAAAAACUCCCUAAGACAGGCCAGAGCCGGCAGCAC 178-E7 43 L-RNAGUGCUGCGGAGUUGAAAACUCCCUAAGACAGGCCAGAGCCGGCAGCAC 178-G6 44 L-RNAGUGCUGCGUAGUGGAAGACUACCUAUGACAGCCUAAUGCUGGCAGCAC 178-A7 45 L-RNAGUGCUGCGGAGUUAAAAACUCCCUAAGACAGGCUAGAGCCGGCAGCAC 178-C7 46 L-RNAGUGCUGCGGCGUGAAAAACGCCCUGCGACUGCCCUUUAUGCAGGCAGCAC 178-E5 47 L-RNAGUGCUGCGUAGUGAAAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 181-F1 48 L-RNAGUGCUGCGUAGUGAAAGACUACCUGUGACAGCCGAAUGCUGGCAGCAC 181-B2 49 L-RNAGUACUGCGUAGUUAAAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 181-C2 50 L-RNAGUGCUGCGUAGUUAAAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 178-A6 51 L-RNAGUGCUGCGUAGUUAAAAACUACCAGCGACAGGCUAGAGCCGGCAGCAC 178-D6 52 L-RNAGUGCUGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCAGCAC 178-D5 53 L-RNAGUGCUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 181-A2 54 L-RNAGGCUGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCAGCC 178-D5-020 55 L-RNAGGCGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCGCC 178-D5-027 56 L-RNAGUGCGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCGCAC 178-D5-030 57 L-RNAGUGCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGCAC 181-A2-002 58 L-RNAGUGCCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGGCAC 181-A2-004 59 L-RNAGUGGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCCAC 181-A2-005 60 L-RNAGUCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGAC 181-A2-006 61 L-RNAUGCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGCA 181-A2-007 62 L-RNAGCUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAGC 181-A2-008 63 L-RNAGCUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAGC 181-A2-011 64 L-RNAGGUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCACC 181-A2-012 65 L-RNAUGGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGC-CA 181-A2-015 66 L-RNAGCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGC 181-A2-016 67 L-RNAGUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAC 181-A2-017 68 L-RNAGG-GCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCCC 181-A2-018 69 L-RNAGAGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCUC 181-A2-019 70 L-RNACGGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCCG 181-A2-020 71 L-RNACCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGG 181-A2-021 72 L-RNACAGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCUG 181-A2-022 73 L-RNACUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAG 181-A2-023 74 L-RNAAGCGUGUUAGUGAAGUGGGUGGCAGGUAAAGGACACGCU 184-B8trc 75 L-RNAAGCGUGGUAGCGGUGUGGGUGGUAGGUAAAGGCCACGCU 184-C6trc 76 L-RNAAGCGUGAUAGAAGAGCGGGUGGUAGGUAAAGGUCAGGCU 184-H5trc 77 L-RNAAGCGUGUUAGGUAGGGUGGUAGUAAGUAAAGGACACGCU 184-A7trc 78 L-RNAAGCGUGUUAGGUGGGUGGUAGUAAGUAAAGGACACGCU 187-A5trc 79 L-RNAAGCGUGUUAGGUGGGUGGUAGUAAGUAAAGGGCACGCU 187-H5trc 80 L-RNACCGCUUAGGUGGGUGGUAGUAAGUAAAGGGGCGG 174-D4-004 81 L-RNAGCGCGAGCAGGUGGGUGGUAGAAUGUAAAGACUCGCGUC 166-A4-002 82 L-RNACGUGUUAGGUGGGUGGUAGUAAGUAAAGGACACG 187-A5trc-001 83 L-RNAGUGUUAGGUGGGUGGUAGUAAGUAAAGGACAC 187-A5trc-002 84 L-RNACGUGUUAGGUGGGUGGUAGUAAGUAAAGGGCACG 187-H5trc-002 85 L-RNAGUGUUAGGUGGGUGGUAGUAAGUAAAGGGCAC 187-H5trc-003 86 L-RNAUGUUAGGUGGGUGGUAGUAAGUAAAGGGCA 187-H5trc-004 87 L-RNAGGACGAGAGUGACAAAUGAUAUAACCUCCUGACUAACGCUGCGGGCGACAGG 177-B3 88 L-RNAGGACCUAUCGCUAAGACAACGCGCAGUCUACGGGACAUUCUCCGCGGACAGG 177-C1 89 L-RNAGGACAAUUGUUACCCCCGAGAGAGACAAAUGAGACAACCUCCUGAAGACAGG 177-C2 90 L-RNAGGACGAAAGUGAGAAAUGAUACAACCUCCUGUUGCUGCGAAUCCGGACAGG 177-E3 91 L-RNAGGACGUAAAAGACGCUACCCGAAAGAAUGUCAGGAGGGUAGACCGACAGG 177-D1 92 L-RNAGGACUAGAAACUACAAUAGCGGCCAGUUGCACCGCGUUAUCAACGACAGG 177-E1 93 L-RNAGGACUAGUCAGCCAGUGUGUAUAUCGGACGCGGGUUUAUUUACUGACAGG 177-A1 94 L-RNAGGACUGUCCGGAGUGUGAAACUCCCCGAGACCGCCAGAAGCGGGGACAGG 177-G3 95 L-RNAGGACUUCUAUCCAGGUGGGUGGUAGUAUGUAAAGAGAUAGAAGUGACAGG 177-C3 96 L-RNAGGACGAGAGCGAACAAUGAUAUAACCUCCUGACGGAAAGAGAUCGACAGG 177-A2 97 L-RNACCUGUGCUACACGCAGUAAGAAGUGAACGUUCAGUAUGUGUGCACAGG 170-E4trc 98 L-RNACGUGAGCCAGGCACCGAGGGCGUUAACUGGCUGAUUGGACACGACACG 166-D2trc 99 L-RNACGUGAACAUGCAAGCUAAGCGGGGCUGUUGGUUGCUUGGCCCGCCACG 174-A2trc 100 L-RNACGUGCAGAGAGAGACCAACCACGUAAAAUCAACCUAAUGGGCCGCACG 174-E2trc 101 L-RNACGUGCAGAGAGAGACCAACCACGUAAAAUCAACCUAAUGGGCCGCACG 183-G3trc 102 L-RNACGUGAACAUUCAAGCUAAGCGGGGCUGUUGGUUGCUUGGCCCGCCACG 183-B2trc 103 L-RNACGUGCCGAGGCGGCGACCAGCGUUACUUAGAGAGGCUUUGGCACCACG 166-B2trc 104 L-RNACGUGAUAACAGCCGUCGGUCAAGAAAACAAAGUUCGGGCGGCGCACG 166-G3trc 105 L-RNACGUGGGUGGCGCACCGAGGGCGAAAAGCCACCAGUAAAGAUAGACCG 166-D1trc 106 L-RNACGUGUGAUCUCCUUUGGGGUGAUUAGCUUAGAGACUUCCCACACG 183-H2trc 107 L-RNAGCACCUUCGCCUAAUACACGUGCCGGCUAGCUAAUACUCGUCCGC 167-A7trc 108 L-RNAGCACGACUUGGGCGACCAGUGAUACUUAGAGAGCAAGUCGUCGGC 167-C7trc 109 L-RNAGCGCGCGCUCAGUAAGAAAUUGAAAGUUCAGAAUGUCGUCGCGC 167-B5trc 110 L-RNAAGUGUGUGGCAGGCUAAGGAGAUAUUCCGAGACCACGCU 184-D7trc 111 L-RNAAGUGUGUGGCAGACUAUGGAUAGACUCCGAGACCACGCU 184-D6trc 112 L-RNAAGCGUGAGGCGACCAGCGGAUUACUUAGAGAGUCACGCU 184-E5trc 113 L-RNAAGCGUGAAGGGGACCAGCGUUACUUACAGAGUUCACGCU 184-G6trc 114 L-RNAAGCGUGUGAUGUAUGUAGCACCGUAUCAGAGGACACGCU 184-B7trc 115 L-RNAAGCGUGAGGCGACCCGUGUUUCGUAGAGAGUCACGCU 184-B6trc 116 L-RNA5′PEG-GCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG NOX-E36-5′PEG 117 L-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG-3′PEG NOX-E36-3′PEG 118 L-RNAGAGAUGGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUUC 188-A3-001119 L-RNA GAUGGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUUC188-A3-004 120 L-RNAGGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUUC 188-A3-005 121L-RNA GGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUU 188-A3-006122 L-RNA GGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCA188-A3-007 = mNOX-E36 123 L-RNAGCUGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCAGC 189-G7-001 124 L-RNACUGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCAG 189-G7-002 125 L-RNAUGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCA 189-G7-003 126 L-RNAGCCGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCGGC 189-G7-007 127 L-RNAGCCGGCUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCGCCGGC 189-G7-008 128 L-RNAGCGCGUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCCGCGC 189-G7-010 129 L-RNAGGGCCUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCGGCCC 189-G7-012 130 D-proteinBiotin- biotinylated humanQPDAINAPVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWV D-MCP-1QDSMDHLDKQTQTPKT 131 D-protein Biotin- biotinylated mouseQPDAVNAPLTCCYSFTSKMIPMSRLESYKRITSSRCPKEAVVFVTKLKREVCADPKKEWV D-MCP-1QTYIKNLDRNQMRSEP-Biotin 132 D-RNAAGCGUGCCCGGAGUGGCAGGGGGACGCGACCUGCAAUAAUGCACGCU 169-B1trc 133 D-RNAAGCGUGCCCGGAGUGGCAGGGGGACGCGACCUGCAAUUGCACGCU 169-F3trc 134 D-RNAAGCGUGCCCGGAGUGGCAGGGGGACGCGACCUGUAAUAAUGCACGCU 169-C1trc 135 D-RNAAGCGUGCCCGGUGUGGCAGGGGGACGCGACCUGCAAUAAUGCGCGCU 169-A3trc 136 D-RNAAGCGUGCCCGGAGUAGCAGGGGGGCGCGACCUGCAAUAAUGCACGCU 169-B2trc 137 D-RNAAGCGUGCCCGGUGUGGUAGGGGGGCGCGAUCUACAAUUGCACGCU 176-B12trc 138 D-RNAAGCGUGCCCGGUGUGACAGGGGGGCGCGACCUGCAUUUGCACGCU 176-D9trc 139 D-RNAAGCGUGCCCGGUGUGGCAGGGGGGCGCGACCUGUAUUUGCACGCU 176-B10trc 140 D-RNAAGCGUGCCCGGAGUGGCAGGGGGGCGCGACCUGCAAUAAUGCACGCU 169-F2trc 141 D-RNAAGCGUGCCCGGUGUGGCAGGGGGGCGCGACCUGCAAUUGCACGCU 176-B9trc 142 D-RNAAGCAUGCCCGGUGUGGCAGGGGGGCGCGACCUGCAUUUGCAUGCU 176-H9trc 143 D-RNAAGCGUGCCCGGUGUGGUAGGGGGGCGCGACCUACAUUUGCACGCU 176-E10trc 144 D-RNAAGUGUGCCAGCUGUGAUGGGGGGGCGCGACCCAUUUUACACACU 176-G9trc 145 D-RNAAGUGUGCCAGCGUGAUGGGGGGGCGCGACCCAUUUUACACACU 176-F9trc 146 D-RNAAGUGUGCGAGCGUGAUGGGGGGGCGCGACCCAUUUUACAUACU 176-C11trc 147 D-RNAAGUGUGCCAGCGUGAUGGGGGGGCGCGACCCAUUUUACAUACU 176-E11trc 148 D-RNAAGUAUGCCAGCGUGAUGGGGGGGCGCGACCCAUUUACAUACU 176-D10trc 149 D-RNAAGUGUGCCAGUGUGAUGGGGGGGCGCGACCCAUUUUACACACU 176-H10trc 150 D-RNAAGCGUGCCAGUGUGAUGGGGGGGCGCGACCCAUUUUACACGCU 176-C9trc 151 D-RNAACGCACGUCCCUCACCGGUGCAAGUGAAGCCGCGGCUCUGCGU 180-B1-001 152 D-RNAACGCACCUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGC 180-A4-002 153 D-RNAACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGU 180-D1-002 154 D-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGU 180-D1-011 155 D-RNAACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGC 180-D1-012 156 D-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGC 180-D1-018 157 D-RNACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCGU 180-D1-034 158 D-RNACGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG 180-D1-035 159 D-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG (D-) 180-D1-036, (D-) NOX-E36160 D-RNA GUGCUGCGUAGUGGAAGACUACCUAAUGACAGCCGAAUGCUGGCAGCAC 178-A8 161D-RNA GUGCUGCGUAGUGGAAGACUACCUAAUGACAGCCUAAUGCUGGCAGCAC 178-F7 162 D-RNAGUGCUGCGUAGUGGAAGACUACCUUAUGACAGCCGAAUGCUGGCAGCAC 178-G7 163 D-RNAGUGCUGCGUAGUGAAAAACUACUGCCAGUGGGUCAGAGCUAGCAGCAC 178-C6 164 D-RNAGUGCUGCGGAGUUAAAAACUCCCUAAGACAGGCCAGAGCCGGCAGCAC 178-E7 165 D-RNAGUGCUGCGGAGUUGAAAACUCCCUAAGACAGGCCAGAGCCGGCAGCAC 178-G6 166 D-RNAGUGCUGCGUAGUGGAAGACUACCUAUGACAGCCUAAUGCUGGCAGCAC 178-A7 167 D-RNAGUGCUGCGGAGUUAAAAACUCCCUAAGACAGGCUAGAGCCGGCAGCAC 178-C7 168 D-RNAGUGCUGCGGCGUGAAAAACGCCCUGCGACUGCCCUUUAUGCAGGCAGCAC 178-E5 169 D-RNAGUGCUGCGUAGUGAAAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 181-F1 170 D-RNAGUGCUGCGUAGUGAAAGACUACCUGUGACAGCCGAAUGCUGGCAGCAC 181-B2 171 D-RNAGUACUGCGUAGUUAAAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 181-C2 172 D-RNAGUGCUGCGUAGUUAAAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 178-A6 173 D-RNAGUGCUGCGUAGUUAAAAACUACCAGCGACAGGCUAGAGCCGGCAGCAC 178-D6 174 D-RNAGUGCUGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCAGCAC 178-D5 175 D-RNAGUGCUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAGCAC 181-A2 176 D-RNAGGCUGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCAGCC 178-D5-020 177 D-RNAGGCGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCGCC 178-D5-027 178 D-RNAGUGCGCGUAGUUAAAAACUACCAGCGACUGGCUAGAGCCGGCGCAC 178-D5-030 179 D-RNAGUGCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGCAC 181-A2-002 180 D-RNAGUGCCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGGCAC 181-A2-004 181 D-RNAGUGGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCCAC 181-A2-005 182 D-RNAGUCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGAC 181-A2-006 183 D-RNAUGCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGCA 181-A2-007 184 D-RNAGCUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAGC 181-A2-008 185 D-RNAGCUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAGC 181-A2-011 186 D-RNAGGUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCACC 181-A2-012 187 D-RNAUGGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGC-CA 181-A2-015 188 D-RNAGCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGC 181-A2-016 189 D-RNAGUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAC 181-A2-017 190 D-RNAGG-GCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCCC 181-A2-018 191 D-RNAGAGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCUC 181-A2-019 192 D-RNACGGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCCG 181-A2-020 193 D-RNACCGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCGG 181-A2-021 194 D-RNACAGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCUG 181-A2-022 195 D-RNACUGCGUAGUGAGAAACUACCAACGACUGGCUAGAGCCGGCAG 181-A2-023 196 D-RNAAGCGUGUUAGUGAAGUGGGUGGCAGGUAAAGGACACGCU 184-B8trc 197 D-RNAAGCGUGGUAGCGGUGUGGGUGGUAGGUAAAGGCCACGCU 184-C6trc 198 D-RNAAGCGUGAUAGAAGAGCGGGUGGUAGGUAAAGGUCAGGCU 184-H5trc 199 D-RNAAGCGUGUUAGGUAGGGUGGUAGUAAGUAAAGGACACGCU 184-A7trc 200 D-RNAAGCGUGUUAGGUGGGUGGUAGUAAGUAAAGGACACGCU 187-A5trc 201 D-RNAAGCGUGUUAGGUGGGUGGUAGUAAGUAAAGGGCACGCU 187-H5trc 202 D-RNACCGCUUAGGUGGGUGGUAGUAAGUAAAGGGGCGG 174-D4-004 203 D-RNAGCGCGAGCAGGUGGGUGGUAGAAUGUAAAGACUCGCGUC 166-A4-002 204 D-RNACGUGUUAGGUGGGUGGUAGUAAGUAAAGGACACG 187-A5trc-001 205 D-RNAGUGUUAGGUGGGUGGUAGUAAGUAAAGGACAC 187-A5trc-002 206 D-RNACGUGUUAGGUGGGUGGUAGUAAGUAAAGGGCACG 187-H5trc-002 207 D-RNAGUGUUAGGUGGGUGGUAGUAAGUAAAGGGCAC 187-H5trc-003 208 D-RNAUGUUAGGUGGGUGGUAGUAAGUAAAGGGCA 187-H5trc-004 209 D-RNAGGACGAGAGUGACAAAUGAUAUAACCUCCUGACUAACGCUGCGGGCGACAGG 177-B3 210 D-RNAGGACCUAUCGCUAAGACAACGCGCAGUCUACGGGACAUUCUCCGCGGACAGG 177-C1 211 D-RNAGGACAAUUGUUACCCCCGAGAGAGACAAAUGAGACAACCUCCUGAAGACAGG 177-C2 212 D-RNAGGACGAAAGUGAGAAAUGAUACAACCUCCUGUUGCUGCGAAUCCGGACAGG 177-E3 213 D-RNAGGACGUAAAAGACGCUACCCGAAAGAAUGUCAGGAGGGUAGACCGACAGG 177-D1 214 D-RNAGGACUAGAAACUACAAUAGCGGCCAGUUGCACCGCGUUAUCAACGACAGG 177-E1 215 D-RNAGGACUAGUCAGCCAGUGUGUAUAUCGGACGCGGGUUUAUUUACUGACAGG 177-A1 216 D-RNAGGACUGUCCGGAGUGUGAAACUCCCCGAGACCGCCAGAAGCGGGGACAGG 177-G3 217 D-RNAGGACUUCUAUCCAGGUGGGUGGUAGUAUGUAAAGAGAUAGAAGUGACAGG 177-C3 218 D-RNAGGACGAGAGCGAACAAUGAUAUAACCUCCUGACGGAAAGAGAUCGACAGG 177-A2 219 D-RNACCUGUGCUACACGCAGUAAGAAGUGAACGUUCAGUAUGUGUGCACAGG 170-E4trc 220 D-RNACGUGAGCCAGGCACCGAGGGCGUUAACUGGCUGAUUGGACACGACACG 166-D2trc 221 D-RNACGUGAACAUGCAAGCUAAGCGGGGCUGUUGGUUGCUUGGCCCGCCACG 174-A2trc 222 D-RNACGUGCAGAGAGAGACCAACCACGUAAAAUCAACCUAAUGGGCCGCACG 174-E2trc 223 D-RNACGUGCAGAGAGAGACCAACCACGUAAAAUCAACCUAAUGGGCCGCACG 183-G3trc 224 D-RNACGUGAACAUUCAAGCUAAGCGGGGCUGUUGGUUGCUUGGCCCGCCACG 183-B2trc 225 D-RNACGUGCCGAGGCGGCGACCAGCGUUACUUAGAGAGGCUUUGGCACCACG 166-B2trc 226 D-RNACGUGAUAACAGCCGUCGGUCAAGAAAACAAAGUUCGGGCGGCGCACG 166-G3trc 227 D-RNACGUGGGUGGCGCACCGAGGGCGAAAAGCCACCAGUAAAGAUAGACCG 166-D1trc 228 D-RNACGUGUGAUCUCCUUUGGGGUGAUUAGCUUAGAGACUUCCCACACG 183-H2trc 229 D-RNAGCACCUUCGCCUAAUACACGUGCCGGCUAGCUAAUACUCGUCCGC 167-A7trc 230 D-RNAGCACGACUUGGGCGACCAGUGAUACUUAGAGAGCAAGUCGUCGGC 167-C7trc 231 D-RNAGCGCGCGCUCAGUAAGAAAUUGAAAGUUCAGAAUGUCGUCGCGC 167-B5trc 232 D-RNAAGUGUGUGGCAGGCUAAGGAGAUAUUCCGAGACCACGCU 184-D7trc 233 D-RNAAGUGUGUGGCAGACUAUGGAUAGACUCCGAGACCACGCU 184-D6trc 234 D-RNAAGCGUGAGGCGACCAGCGGAUUACUUAGAGAGUCACGCU 184-E5trc 235 D-RNAAGCGUGAAGGGGACCAGCGUUACUUACAGAGUUCACGCU 184-G6trc 236 D-RNAAGCGUGUGAUGUAUGUAGCACCGUAUCAGAGGACACGCU 184-B7trc 237 D-RNAAGCGUGAGGCGACCCGUGUUUCGUAGAGAGUCACGCU 184-B6trc 238 D-RNA5′PEG-GCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG NOX-E36-5′PEG 239 D-RNAGCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG-3′PEG NOX-E36-3′PEG 240 D-RNAGAGAUGGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUUC 188-A3-001241 D-RNA GAUGGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUUC188-A3-004 242 D-RNAGGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUUC 188-A3-005 243D-RNA GGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCAUU 188-A3-006244 D-RNA GGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCA(D-)188-A3-007 = (D-) mNOX-E36 245 D-RNAGCUGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCAGC 189-G7-001 246 D-RNACUGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCAG 189-G7-002 247 D-RNAUGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCA 189-G7-003 248 D-RNAGCCGGUUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCACCGGC 189-G7-007 249 D-RNAGCCGGCUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCGCCGGC 189-G7-008 250 D-RNAGCGCGUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCCGCGC 189-G7-010 251 D-RNAGGGCCUACCGAGGGGGCGUCGUUGGAGUUUGGUUGGUUGUCGGCCC 189-G7-012 252 L-proteinQPDAVNAPLTCCYSFTGKMIPMSRLENYKRITSSRCPKEAVVFVTKLKREICADPNKEWVQ rat MCP-1KYIRKLDQNQVRSET 253 L-RNA5′PEG-GGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCA mNOX-E36-5′PEG254 L-RNA GGCGACAUUGGUUGGGCAUGAGGCGAGGCCCUUUGAUGAAUCCGCGGCCA-3′PEGmNOX-E36-3′PEG 255 L-DNA 5′-GAGGGACGTGC-(Spacer18)₂-NH4⁺-3′NOX-E36 Capture probe 256 L-DNA 5′-Biotin-(Spacer18)₂-CGCAGAGCCNOX-E36 Detect (-ion) probe 257 L-ProteinKSMQVPFSRCCFSFAEQEIPLRAILCYRNTSSICSNEGLIFKLKRGKEACALDTVGWVQRHRKMCCL1/I-309 LRHCPSKRK 258 L-ProteinSLAADTPTACCFSYTSRQIPQNFIADYFETSSQCSKPGVIFLTKRSRQVCADPSEEWVQKYVSDCCL3/MIP-1α LELSA 259 L-ProteinAPMGSDPPTACCFSYTARKLPRNFVVDYYETSSLCSQPAVVFQTKRSKQVCADPSESWVQEYVYCCL4/MIP-1β DLELN 260 L-ProteinSPYSSDTTPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINSCCL5/RANTES LEMS 261 L-ProteinFNPQGLAQPDALNVPSTCCFTFSSKKISLQRLKSYVITTSRCPQKAVIFRTKLGKEICADPKEKCCL13/MCP-4 WVQNYMKHLGRKAHTLKT 262 L-ProteinTKTESSSRGPYHPSECCFTYTTYKIPRQRIMDYYETNSQCSKPGIVFITKRGHSVCTNPSDKWVCCL14/HCC-1 QDYIKDMKEN 263 L-ProteinASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPASPIVKKIIECXCL1/GROα KMLNSDKSN 264 L-ProteinAPLATELRCQCLQTLQGIHLKNIQSVKVKSPGPHCAQTEVIATLKNGQKACLNPASPMVKKIIECXCL2/GROβ KMLKNGKSN 265 L-ProteinASVVTELRCQCLQTLQGIHLKNIQSVNVRSPGPHCAQTEVIATLKNGKKACLNPASPMVQKIIECXCL3/GROγ KILNKGSTN 266 L-ProteinEAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIICXCL4/PF4 KKLLES 267 L-ProteinGPAAAVLRELRCVCLQTTQGVHPKMISNLQVFAIGPQCSKVEVVASLKNGKEICLDPEA CXCL5/ENA-78PFLKKVIQKILDGGNKEN 268 L-ProteinGPVSAVLTELRCTCLRVTLRVNPKTIGKLQVFPAGPQCSKVEVVASLKNGKQVCLDPEA CXCL6/GCP-2PFLKKVIQKILDSGNKKN 269 L-ProteinSSTKGQTKRNLAKGKEESLDSDLYAELRCMCIKTTSGIHPKNIQSLEVIGKGTHCNQVE CXCL7/NAP-2VIATLKDGRKICLDPDAPRIKKIVQKKLAGDESAD 270 L-ProteinEGAVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLD CXCL8/IL-8PKENWVQRVVEKFLKRAENS 271 L-ProteinTPVVRKGRCSCISTNQGTIHLQSLKDLKQFAPSPSCEKIEIIATLKNGVQTCLNPDSAD CXCL9/MIGVKELIKKWEKQVSQKKKQKNGKKHQKKKVLKVRKSQRSRQKKTT 272 L-ProteinVPLSRTVRCTCISISNQPVNPRSLEKLEIIPASQFCPRVEIIATMKKKGEKRCLNPESK CXCL10/IP-10AIKNLLKAVSKERSKRSP 273 L-ProteinFPMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSK CXCL11/I-TACQARLIIKKVERKNF 274 L-ProteinKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQCXCL12α/SDF-1αa EYLEKALNKRFKM 275 L-ProteinKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQCXCL12β/SDF-1β EYLEKALNKRFKM 276 L-ProteinQHHGVTKCNITCSKMTSKIPVALLIHYQQNQASCGKRAIILETRQHRLFCADPKEQWVKCX₃CL1/Fractalkine DAMQHLDRQAAALTRNG 277 L-ProteinVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVXCL1/Lymphotactin VRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG 278 L-RNA5′-Biotin-GCACGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUCUGCG biotinylated NOX-E36279 L-RNA 5′-UAAGGAAACUCGGUCUGAUGCGGU AGCGCUGUGCAGAGCU POC 280 L-RNA5′-PEG-UAAGGAAACUCGGUCUGAUGCGGU AGCGCUGUGCAGAGCU-3′ POC-PEG 281 L-DNA5′-CCAATGTCGCC-(Spacer18)₂-NH4⁺-3′ mNOX-E36 Capture probe 282 L-DNA5′-Biotin-(Spacer18)₂-CGCAGAGCC mNOX-E36 Detect (-ion) probe 283L-protein QPDAINSPVTCCYTFTGKKISSQRLGSYKRVTSSKCPKEAVIFKTILAKEIChorse MCP-1 (Equus ADPEQKWVQDAVKQLDKKAQTPKP caballus) 284 L-proteinQPDAINSQVACCYTFNSKKISMQRLMNYRRVTSSKCPKEAVIFKTILGKELC bovine MCP-1 (BosADPKQKWVQDSINYLNKKNQTPKP Taurus) 285 L-proteinQPDAVNAPLTCCYSFTGKMIPMSRLENYKRITSSRCPKEAVVFVTKLKREIC rat MCP-1 (RattusADPNKEWVQKYIRKLDQNQVRSETTVFYKIASTLRTSAPLNVNLTHKSEANA norvegicus)STLFSTTTSSTSVEVTSMTEN

The present invention is further illustrated by the figures, examplesand the sequence listing from which further features, embodiments andadvantages may be taken, wherein

FIG. 1 shows an alignment of sequences of related RNA ligands binding tohuman MCP-1 indicating the sequence motif (“Type 1A”) that is in apreferred embodiment in its entirety essential for binding to humanMCP-1;

FIG. 2 shows an alignment of sequences of related RNA ligands binding tohuman MCP-1 indicating the sequence motif (“Type 1B”) that is in apreferred embodiment in its entirety essential for binding to humanMCP-1 and derivatives of RNA ligands 180-D1-002;

FIG. 3 shows an alignment of sequences of related RNA ligands binding tohuman MCP-1 indicating the sequence motif (“Type 2”) that is in apreferred embodiment in its entirety essential for binding to humanMCP-1;

FIG. 4 shows an alignment of sequences of related RNA ligands binding tohuman MCP-1 indicating the sequence motif (“Type 3”) that is in apreferred embodiment in its entirety essential for binding to humanMCP-1;

FIG. 5 shows derivatives of RNA ligands 178-D5 and 181-A2 (human MCP-1RNA ligands of sequence motif “Type 3”):

FIG. 6 shows an alignment of sequences of related RNA ligands binding tohuman MCP-1 indicating the sequence motif (“Type 4”) that is in apreferred embodiment in its entirety essential for binding to humanMCP-1 (other sequences);

FIG. 7 shows a table of sequences of several different RNA ligandsbinding to human MCP-1 which can not be related to the MCP-1 bindingsequence motifs “Type 1A”, “Type 1B”; “Type 2”, “Type 3” or “Type 4”;

FIG. 8 shows alignments of derivatives of RNA ligand 188-A3-001 and of189-G7-001 that bind to murine MCP-1;

FIG. 9 shows the result of a binding analysis of the aptamer D-NOX-E36to biotinylated human D-MCP-1 at room temperature and 37° C.,represented as binding of the aptamer over concentration of biotinylatedhuman D-MCP-1;

FIG. 10 shows the result of a binding analysis of the aptamer D-mNOX-E36to biotinylated murine D-MCP-1 at 37° C., represented as binding of theaptamer over concentration of biotinylated murine D-MCP-1;

FIG. 11 shows MCP-1-induced Ca⁺⁺-release in THP-1 cells, whereas adose-response curve for human MCP-1 was obtained, indicating a halfeffective concentration (EC₅₀) of approximately 3 nM, represented asdifference in fluorescence to blank over concentration of human MCP-1;

FIG. 12 shows the efficacy of Spiegelmer NOX-E36 in a calcium releaseassay; cells were stimulated with 3 nM human MCP-1 preincubated at 37°C. with various amounts of Spiegelmer NOX-E36, represented as percentageof control over concentration of NOX-E36;

FIG. 13 shows the efficacy of Spiegelmer mNOX-E36 in a calcium releaseassay; cells were stimulated with 5 nM murine MCP-1 preincubated at 37°C. with various amounts of Spiegelmer mNOX-E36, represented aspercentage of control over concentration of mNOX-E36:

FIG. 14 shows the human MCP-1-induced chemotaxis of THP-1 cells whereasafter 3 hours migration of THP-1 cells towards various MCP-1concentrations a dose-response curve for MCP-1 was obtained, representedas X-fold increase compared to control over concentration of humanMCP-1;

FIG. 15 shows the efficacy of Spiegelmer NOX-E36 in a chemotaxis assay;cells were allowed to migrate towards 0.5 nM human MCP-1 preincubated at37° C. with various amounts of Spiegelmer NOX-E36, represented aspercentage of control over concentration of Spiegelmer NOX-E36;

FIG. 16 shows the efficacy of Spiegelmer mNOX-E36 in a chemotaxis assay;cells were allowed to migrate towards 0.5 nM murine MCP-1 preincubatedat 37° C. with various amounts of Spiegelmer NOX-E36, represented aspercentage of control over concentration of Spiegelmer mNOX-E36;

FIG. 17 shows the Biacore 2000 sensorgram indicating the K_(D) value ofSpiegelmer NOX-E-36 binding to human MCP-1 which was immobilized on aPioneerF1 sensor chip by amine coupling procedure, represented asresponse (RU) over time;

FIG. 18 shows the Biacore 2000 sensorgram indicating binding ofSpiegelmer NOX-E36 to human MCP-family proteins (huMCP-1, huMCP-2,huMCP-3) and human eotaxin, which were immobilized by amine couplingprocedure on a PioneerF1 and a CM4 sensor chip, respectively,represented as response (RU) over time;

FIG. 19 shows the Biacore 2000 sensorgram indicating binding ofSpiegelmer NOX-E36 to MCP-1 from different species (canine MCP-1, monkeyMCP-1, human MCP-1, porcine MCP-1, rabbit MCP-1, mouse MCP-1, rat MCP-1)whereas different forms of MCP-1 were immobilized by amine couplingprocedure on PioneerF1 and a CM4 sensor chips, respectively, representedas response (RU) over time;

FIG. 20 shows the Biacore 2000) sensorgram indicating the K_(D) value ofSpiegelmer 181-A2-018 binding to human MCP-1 which was immobilized on aCM4 sensor Chip by amine coupling procedure, represented as response(RU) over time;

FIG. 21 shows the Biacore 2000 sensorgram indicating binding ofSpiegelmer 181-A2-018 to human MCP-family proteins (huMCP-1, huMCP-2,huMCP-3) and human eotaxin which were immobilized by amine couplingprocedure on a PioneerF1 and a CM4 sensor chip, respectively,represented as response (RU) over time;

FIG. 22 shows the Biacore 2000 sensorgram indicating binding ofSpiegelmer 181-A2-018 to MCP-1 from different species (canine MCP-1,monkey MCP-1, human MCP-1, porcine MCP-1, rabbit MCP-1, mouse MCP-1, ratMCP-1) whereas different forms of MCP-1 were immobilized by aminecoupling procedure on PioneerF1 and a CM4 sensor chips, respectively,represented as response (RU) over time:

FIG. 23 shows a Clustal W alignment of MCP-1 from different mammalianspecies as well as human MCP-2, MCP-3, and eotaxin (Positions 1-76only);

FIG. 24A shows a table summarizing the binding specificity of NOX-E36and 181-A24-18 regarding MCP-1 from different mammalian species as wellas human MCP-2. MCP-3, and eotaxin;

FIG. 24B shows a table summarizing the selectivity of NOX-E36 asdetermined by Biacore analysis whereby biotinylated NOX-E36 wasimmobilized on a sensor chip surface and binding of a panel of variousCC and CXC chemokines to NOX-E36 was analyzed;

FIG. 24C shows the kinetic analysis of NOX-E36 interacting withchemokines as determined by Biacore analysis whereby the chemokines wereimmobilized covalently on a CM5 sensor chip surface and variousconcentrations of the NOX-E36 were injected and NOX-E36s bindingbehaviour was analyzed using the BiaEvaluation software:

FIG. 24D shows the chemotaxis dose-response curve of THP-1 cellstimulation with MIP-1α with a half-effective concentration of about 0.2nM;

FIG. 24E shows the Inhibition of MIP-1α induced chemotaxis by NOX-E36,NOX-E36 had no influence on the MIP1a induced chemotaxis of THP-1 cells;

FIG. 25 shows the efficacy of Spiegelmer NOX-E36-3′-PEG in a calciumrelease assay; cells were stimulated with 3 nM human MCP-1 preincubatedat 37°C with various amounts of Spiegelmer NOX-E36-3′-PEG, representedas percentage of control over concentration of SpiegelmerNOX-E36-3′-PEG;

FIG. 26 shows the efficacy of Spiegelmer NOX-E36-3′-PEG in a chemotaxisassay; cells were allowed to migrate towards 0.5 nM human MCP-1preincubated at 37° C. with various amounts of SpiegelmerNOX-E36-3′-PEG, represented as percentage of control over concentrationof NOX-E36-3′-PEG;

FIG. 27A shows the efficacy of Spiegelmer NOX-E36-5′-PEG in a calciumrelease assay; cells were stimulated with 3 nM human MCP-1 preincubatedat 37° C. with various amounts of Spiegelmer NOX-E36-5′-PEG, representedas percentage of control over concentration of SpiegelmerNOX-E36-5′-PEG;

FIG. 27B shows the efficacy of Spiegelmer NOX-E36-5′-PEG in a chemotaxisassay, cells were allowed to migrate towards 0.5 nM human MCP-1preincubated at 37° C. with various amounts of SpiegelmerNOX-E36-5′-PEG, represented as percentage of control over concentrationof Spiegelmer NOX-E36-5′-PEG;

FIG. 28 shows murine MCP-1-induced Ca⁺⁺-release in THP-1 cells, whereasa dose-response curve for murine MCP-1 was obtained, indicating a halfeffective concentration (EC₅₀) of approximately 5 nM, represented asdifference in fluorescence to blank over concentration of murine MCP-1;

FIG. 29 shows the efficacy of anti-murine MCP-Spiegelmer mNOX-E36-3′-PEGin a calcium release assay; cells were stimulated with 3 nM murine MCP-1preincubated at 37° C. with various amounts of SpiegelmermNOX-E36-3′-PEG, represented as percentage of control over concentrationof Spiegelmer mNOX-E36-3′-PEG:

FIG. 30 shows the murine MCP-1-induced chemotaxis of THP-1 cells whereasafter 3 hours migration of THP-1 cells towards various mMCP-1concentrations a dose-response curve for mMCP-1 was obtained,represented as X-fold increase compared to control over concentration ofmurine MCP-1;

FIG. 31 shows the efficacy of anti-murine MCP-1 SpiegelmermNOX-E36-3′-PEG in a chemotaxis assay, cells were allowed to migratetowards 0.5 nM murine MCP-1 preincubated at 37° C. with various amountsof Spiegelmer mNOX-E36-3′-PEG, represented as percentage of control overconcentration of anti-murine Spiegelmer mNOX-E36-3′-PEG:

FIG. 32 shows the Biacore 2000 sensorgram indicating the K_(D) value ofaptamer D-mNOX-E36 binding to murine D-MCP-1 which was immobilized on aPioneerF1 sensor chip by amine coupling procedure, represented asresponse (RU) over time;

FIG. 33 shows the Biacore 2000 sensorgram indicating binding of aptamerD-mNOX-E36 to human D-MCP-L and murine D-MCP-1 whereas the two differentforms of D-MCP-1 were immobilized by amine coupling procedure onPioneerF1 and a CM4 sensor chips, respectively, represented as response(RU) over time;

FIGS. 34A-34I show renal sections of 24-week old MRL^(lpr/lpr) mice,stained with periodic acid Schiff (PAS), antibodies for Mac-2(macrophages) and CD3 (T cells) as indicated; images are representativefor 7-12 mice in each group (original magnification PAS: ×100, PASinserts: ×400, Mac2: ×400, CD3: ×100:

FIG. 35 shows a table illustrating renal function parameters andhistological findings in the different groups of 24-week oldMRL^(lpr/lpr) mice:

FIG. 36 shows the quantification of histological changes by morphometryperformed on silver stained sections of mice from all groups; A,interstitial volume index; B, tubular dilation index, and C, tubularcell damage index were calculated as percentage of high power field andare expressed as means±SEM:

FIG. 37 shows the survival of MRL^(lpr/lpr) mice of the varioustreatment groups as calculated by Kaplan-Meier analysis;

FIGS. 38A & 38B show renal mRNA expression for the CC-chemokines CCL2and CCL5 as determined by real-time RT-PCR using total renal RNA pooledfrom 5 mice of each group whereby RNA levels for each group of mice areexpressed per respective 18S rRNA expression;

FIGS. 39A & 39B show reduction of lung pathology by treatment withmNOX-E36-3′PEG; lung tissue was prepared from of all groups at age 24weeks and scored semiquantitatively; treatment with mNOX-E36 andmNOX-E36-3′PEG reduced peribronchiolar inflammation in MRL^(lpr/lpr)mice; images are representative for 7-11 mice in each group; originalmagnification ×100;

FIG. 40 shows cutaneous lupus manifestations of MRL^(lpr/lpr) mice atage 24 weeks which typically occur at the facial or neck area (leftmouse) which were less common in anti-mCCL2 Spiegelmer-treated mice(right mouse);

FIG. 41 shows serum and histological findings in MRL^(lpr/lpr) mice atage 24 weeks;

FIG. 42 shows the pharmacokinetics of pegylated and unpegylatedanti-mCCL2 Spiegelmers in plasma during the study, indicated as plasmaconcentration of Spiegelmer mNOX-E36 as a function of time;

FIG. 43 shows flow cytometry for CCR2 on bone marrow and peripheralblood in 24 week old vehicle- or mNOX-E36-3′PEG-treated MRL^(lpr/lpr)mice; data are shown as mean percentage of CCR2 positive cells±SEM ineither bone marrow or peripheral blood in 5 mice of each group;

FIG. 44 shows serum CCL2 levels in PoC-PEG- (white bars) andmNOX-E36-3′PEG (mNOX-E36-P)-treated (black bars) 1K db/db mice asdetermined by ELISA at different time points as indicated; data aremeans±SEM; *, p<0.05 mNOX-E36-3′PEG (mNOX-E36-P) vs. PoC-PEG:

FIG. 45 shows the infiltrated number of Mac-2 and Ki-67 positive cellsin the glomeruli and the interstitium of untreated or POC-PEG or rathermNOX-E36-3′PEG treated db/db mice;

FIG. 46 shows the diabetic glomerulosclerosis in 6 months old db/dbmice; renal sections from mice of the different groups were stained withperiodic acid Schiff and 15 glomeruli from each renal section werescored for the extent of glomerulosclerosis; images show representativeglomeruli graded to the respective scores as indicated, originalmagnification 400×; the graph illustrates the mean percentage of eachscore±SEM from all mice in each group (n=7-10); *, p<0.05 formNOX-E36-3′PEG (mNOX-E36-P) vs. PoC-PEG (PoC-P)-treated 1K db/db mice;

FIG. 47 shows the glomerular filtration rate (GFR) in 6 months oldmNOX-E36-3′PEG (mNOX-E36-P)- and PoC-PEG(PoC-P)-treated 1K db/db mice;GFR was determined by FITC-inulin clearance kinetics in the groups ofPoC-PEG- and mNOX-E36-3′PEG-treated 1K db/db mice at the end of thestudy;

FIG. 48 shows tubular atrophy and interstitial volume of 6 months olddb/db mice; images of silver-stained renal sections illustraterepresentative kidneys from the respective groups (originalmagnification 100×); values represent means±SEM of the respectivemorphometric analysis index from 7-10 mice in each group; *, p<0.05 2Kdb/db vs. BKS wild-type mice; ^(#), p<0.05 1K vs. 2K db/db mice; ^(†),p<0.05 mNOX-E36-3′PEG (mNOX-E36-PEG)- vs. PoC-PEG-treated 1K db/db mice;

FIG. 49 shows renal CCL2 mRNA expression db/db mice as determined byreal-time RT-PCR using total renal RNA pooled from 6-10 mice of eachgroup; mRNA levels for each group of mice are expressed per respective18 S rRNA expression; and

FIG. 50 shows spatial CCL2 expression in kidneys of db/db mice asdetermined by immunostaining; images illustrate representative sectionsof kidneys from 6 months old mice of the respective groups as indicated(original magnification, 200×).

EXAMPLE 1 Nucleic Acids that Bind Human MCP-1

Using biotinylated human D-MCP-1 as a target, several nucleic acids thatbind to human MCP-1 could be generated the nucleotide sequences of whichare depicted in FIGS. 1 through 7. The nucleic acids were characterizedon the aptamer, i. e. D-nucleic acid level using competitive or directpull-down assays with biotinylated human D-MCP-1 (Example 4) or on theSpiegelmer level, i. e. L-nucleic acid with the natural configuration ofMCP-1 (L-MCP) by surface plasmon resonance measurement using a Biacore2000 instrument (Example 7), an in vitro cell culture Ca⁺⁺-release assay(Example 5), or an in vitro chemotaxis assay (Example 6).

The nucleic acid molecules thus generated exhibit different sequencemotifs, four main types are defined in FIGS. 1 and 2 (Type 1A/1B), FIG.3 (Type 2), FIGS. 4 and 5 (Type 3), and FIG. 6 (Type 4). AdditionalMCP-1 binding nucleic acids which can not be related to each other andto the different sequence motifs described herein, are listed in FIG. 7.For definition of nucleotide sequence motifs, the IUPAC abbreviationsfor ambiguous nucleotides is used:

S strong G or C; W weak A or U; R purine G or A; Y pyrimidine C or U; Kketo G or U; M imino A or C; B not A C or U or G; D not C A or G or U; Hnot G A or C or U; V not U A or C or G; N all A or G or C or U

If not indicated to the contrary, any nucleic acid sequence or sequenceof stretches and boxes, respectively, is indicated in the 5′→3′direction.

Type 1A MCP-1 Binding Nucleic Adds (FIG. 1)

As depicted in FIG. 1 all sequences of MCP-1 binding nucleic acids ofType 1A comprise several sequences stretches or boxes whereby boxes

and

are the 5′- and 3′ terminal stretches that can hybridize with eachother. However, such hybridization is not necessarily given in themolecule as actually present under physiological conditions. Boxes B2,B3, B4,

and box B6 are flanked by box

and box

.

The nucleic acids were characterized on the aptamer level using directand competitive pull-down assays with biotinylated human D-MCP-1 inorder to rank them with respect to their binding behaviour (Example 4).Selected sequences were synthesized as Spiegelmer (Example 3) and weretested using the natural configuration of MCP-1 (L-MCP) in an in vitrocell culture Ca⁺⁺-release assay (Example 5).

The sequences of the defined boxes may be different between the MCP-1binding nucleic acids of Type 1A which influences the binding affinityto MCP-1. Based on binding analysis of the different MCP-1 bindingnucleic acids summarized as Type 1A MCP-1 binding nucleic acids, theboxes

, B2, B3, B4,

B6 and

and their nucleotide sequences as described in the following areindividually and more preferably in their entirety essential for bindingto MCP-1:

-   -   boxes        and        are the 5′- and 3′ terminal stretches can hybridize with each        other; where        is        , preferably        ; and where        is        , preferably        ,    -   box B2, which is CCCGGW, preferably CCCGGU;    -   box B3, which is GUR, preferably GUG;    -   box B4, which is RYA, preferably GUA;    -   box        , which is        , preferably        ;    -   box B6, which is UGCAAUAAUG or URYAWUUG, preferably UACAUUUG;

As depicted in FIG. 1, the nucleic acid molecule referred to as176-E10trc has the best binding affinity to MCP-1 (as aptamer in thepull-assay with a K_(D) of 5 nM as well as Spiegelmer with an IC₅₀ of4-5 nM in in vitro cell culture Ca⁺⁺-release assay) and therefore mayconstitute the optimal sequence and the optimal combination of sequenceelements

, B2, B3, B4,

B6 and

.

Type 1B MCP-1 Binding Nucleic Acids (FIG. 2)

As depicted in FIG. 2, all sequences of Type 1B comprise severalsequences stretches or boxes whereby boxes

and

are the 5′- and 3′ terminal stretches that can hybridize with each otherand boxes B2, B3, B4,

and box B6 are flanked by box

and box

. However, such hybridization is not necessarily given in the moleculeas actually present under physiological conditions.

The nucleic acids were characterized on the aptamer level using directand competitive pull-down assays with biotinylated human D-MCP-1 inorder to rank them with respect to their binding behaviour (Example 4).Selected sequences were synthesized as Spiegelmer (Example 3) and weretested using the natural configuration of MCP-1 (L-MCP) in an in vitrocell culture Ca⁺⁺-release assay (Example 5).

The sequences of the defined boxes may be different between the MCP-1binding nucleic acids of Type 1B which influences the binding affinityto MCP-1. Based on binding analysis of the different MCP-1 bindingnucleic acids summarized as Type B MCP-1 binding nucleic acids, theboxes

, B2, B3, B4,

B6 and

and their nucleotide sequences as described in the following areindividually and more preferably in their entirety essential for bindingto MCP-1:

-   -   boxes        and        that can hybridize with each other; where        is        , preferably        ; and where        is        , preferably        ;    -   box B2, which is CCAGCU or CCAGY, preferably CCAGU;    -   box B3, which is GUG;    -   box B4, which is AUG;    -   box        , which is        ;    -   box B6, which is CAUUUUA or CAUUUA, preferably CAUUUUA;

As depicted in FIG. 2, the nucleic acid referred to as 176-C9trc has thebest binding affinity to MCP-1 (as aptamer in the pull-down assay with aK_(D) of 5 nM as well as Spiegelmer with an IC₅₀ of 4-5 nM in in vitrocell culture Ca⁺⁺-release assay) and therefore may constitute theoptimal sequence and the optimal combination of sequence elements

, B2, B3, B4,

B6 and

.

Type 2 MCP-1 Binding Nucleic Acids (FIG. 3)

As depicted in FIG. 3, all sequences of Type 2 comprise severalsequences stretches or boxes whereby boxes

and

are the 5′- and 3′ terminal stretches that can hybridize with each otherand box B2 is the central sequence element. However, such hybridizationis not necessarily given in the molecule as actually present underphysiological conditions.

The nucleic acids were characterized on the aptamer level using directand competitive pull-down assays with biotinylated human D-MCP-1 inorder to rank them with respect to their binding behaviour (Example 4).Selected sequences were synthesized as Spiegelmer (Example 3) and weretested using the natural configuration of MCP-1 (L-MCP) in vitro cellculture Ca⁺⁺-release (Example 5) or in vitro chemotaxis assays (Example6).

The sequences of the defined boxes may be different between the MCP-1binding nucleic acids of Type 3 which influences the binding affinity toMCP-1. Based on binding analysis of the different MCP-1 binding nucleicacids summarized as Type 2 MCP-1 binding nucleic acids, the boxes

, B2, and

and their nucleotide sequences as described in the following areindividually and more preferably in their entirety essential for bindingto MCP-1:

-   -   boxes        and        , 5′- and 3′ terminal stretches that can hybridize with each        other; where        is        and        is        , or        is        and        is        , or        is        and        is        or        ; preferably        is        and        is        ;    -   box B2,

CSUCCCUCACCGGUGCAAGUGAAGCCGYGGCUC,

-   -    preferably

CGUCCCUCACCGGUGCAAGUGAAGCCGUGGCUC

As depicted in FIG. 3, the nucleic acid referred to as 180-D1-002 aswell as the derivatives of 180-D1-002 like 180-D1-011, 180-D1-012,180-D1-035, and 180-D1-036 (=NOX-E36) have the best binding affinity toMCP-1 as aptamer in the pull-down or competitive pull-down assay with anK_(D) of <1 nM and therefore may constitute the optimal sequence and theoptimal combination of sequence elements

, B2, and

.

For nucleic acid molecule D-NOX-E36 (D-180-D1-036; SEQ. ID No. 159), adissociation constant (K_(D)) of 890±65 pM at room temperature (RT) andof 146±13 pM at 37° C. was determined (Example 4; FIG. 9). Therespective Spiegelmer NOX-E36 (180-D1-036; SEQ. ID No. 37) exhibited aninhibitory concentration (IC₅₀) of 3-4 nM in an in vitro Ca⁺⁺-releaseassay (Example 5; FIG. 12) and of ca. 0.5 nM in an in vitro chemotaxisassay (Example 6; FIG. 15). For the PEGylated derivatives of NOX-E36,NOX-E36-3′PEG and NOX-E36-5′PEG, IC₅₀s of ca. 3 nM were determined inthe Ca⁺⁺-release assay (Example 5, FIG. 25 and FIG. 27A) and <1 nM inthe chemotaxis assay (Example 6; FIG. 26 and FIG. 27B).

Type 3 MCP-1 Binding Nucleic Acids (FIGS. 4+5)

As depicted in FIGS. 4 and 5, all sequences of Type 3 comprise severalsequence stretches or boxes whereby three pairs of boxes arecharacteristic for Type 3 MCP-1 binding nucleic acids. Both boxes

and

as well as boxes B2A and B2B, as well as boxes B5A and B5B bear theability to hybridize with each other. However, such hybridization is notnecessarily given in the molecule as actually present underphysiological conditions. Between these potentially hybridized sequenceelements, non-hybridizing nucleotides are located, defined as box B3,box B4 and box

.

The nucleic acids were characterized on the aptamer level using directand competitive pull-down assays with biotinylated human D-MCP-1 inorder to rank them with respect to their binding behavior (Example 4).Selected sequences were synthesized as Spiegelmer (Example 3) and weretested using the natural configuration of MCP-1 (L-MCP) in in vitrochemotaxis assays (Example 6) or via Biacore measurements (Example 7).

The sequences of the defined boxes may be different between the MCP-1binding nucleic acids of Type 3 which influences the binding affinity toMCP-1. Based on binding analysis of the different MCP-1 binding nucleicacids summarized as Type 3 MCP-1 binding nucleic acids, the boxes

, B2A, B3, B2B, B4, B5A,

, B5B,

and their nucleotide sequences as described in the following areindividually and more preferably in their entirety essential for bindingto MCP-1:

-   -   boxes        and        , 5′- and 3′ terminal stretches that can hybridize with each        other; where        is        and        is        ; preferably        is        and        is        ;    -   or        is        and        is        ; preferably        is        and        is        ;    -   or        is        and        is        ; preferably        is        and        is        ;    -   or        is        and        is        ; preferably        is        and        is        ; most preferably        is        and        is        ;    -   boxes B2A and B2B, stretches that can hybridize with each other;        where B2A is GKMGUand B2B is ACKMC; preferably B2A is GUAGU and        B2B is ACUAC;    -   box B3, which is KRRAR, preferably UAAAA or GAGAA;    -   box B4, which is CURYGA or CUWAUGA or CWRMGACW or UGCCAGUG,        preferably CAGCGACU or CAACGACU;    -   B5A and B5B, stretches that can hybridize with each other; where        B5A is GGY and B5B is GCYR whereas GCY can hybridize with the        nucleotides of B5A; or B5A is CWGC and B5B is GCRWG; preferably        BA is GGC and B5B is GCCG;    -   box        which is:        or        or        , preferably        .

As depicted in FIGS. 4 and 5, the nucleic acid referred to as 178-D5 andits derivative 178-D5-030 as well as 181-A2 with its derivatives181-A2-002, 181-A2-004, 181-A2-005, 181-A2-006, 181-A2-007, 181-A2-017,181-A2-018, 181-A2-019, 181-A2-020, 181-A2-021, and 181-A2-023 have thebest binding affinity to MCP-1. 178-D5 and 178-D5-030 were evaluated asaptamers in direct or competitive pull-down assays (Example 4) with anK_(D) of approx. 500 pM. In the same experimental set-up, 181-A2 wasdetermined with an K_(D) of approx. 100 pM. By Biacore analysis (Example7), the K_(D) of 181-A2 and its derivatives towards MCP-1 was determinedto be 200-300 pM. In Ca⁺⁺ release and chemotaxis assays with culturedcells (Example 5 and 6, respectively), for both 178-D5 and 181-A2, anIC₅₀ of approx. 500 pM was measured. Therefore, 178-D5 as well as 181-A2and their derivatives may constitute the optimal sequence and theoptimal combination of sequence elements

, B2A, B3, B2B, B4, B5A,

, B5B and

.

Type 4 MCP-1 Binding Nucleic Acids (FIG. 6)

As depicted in FIG. 6, all sequences of Type 4 comprise severalsequences, stretches or boxes whereby boxes

and

are the 5′- and 3′ terminal stretches that can hybridize with each otherand box B2 is the central sequence element.

The nucleic acids were characterized on the aptamer level using directpull-down assays with biotinylated human D-MCP-1 in order to rank themwith respect to their binding behavior (Example 4). Selected sequenceswere synthesized as Spiegelmer (Example 3) and were tested using thenatural configuration of MCP-1 (L-MCP) in an in vitro cell cultureCa⁺⁺-release (Example 5) and/or chemotaxis assay (Example 6).

The sequences of the defined boxes may differ among the MCP-1 bindingnucleic acids of Type 4 which influences the binding affinity to MCP-1.Based on binding analysis of the different MCP-1 binding nucleic acidsummarized as Type 4 MCP-1 binding nucleic acids, the boxes

, B2, and

and their nucleotide sequences as described in the following areindividually and more preferably in their entirety essential for bindingto MCP-1:

-   -   boxes        and        , 5′- and 3′ terminal stretches that can hybridize with each        other; where        is        and        is        ; or        is        and        is        ; or        is        and        is        ; or        is        and        is        or        is        and        is        ; preferably        is        and        is        ; mostly preferred B1A is        and        is        ; and    -   box B2, which is

AGNDRDGBKGGURGYARGUAAAG

-   -    or

AGGUGGGUGGUAGUAAGUAAAG

-   -    or

CAGGUGGGUGGUAGAAUGUAAAGA,

-   -    preferably

AGGUGGGUGGUAGUAAGUAAAG

As depicted in FIG. 6, the nucleic acid referred to as 174-D4-004 and166-A4-002 have the best binding affinity to MCP-1 (as Spiegelmer withan IC₅₀ of 2-5 nM in in vitro cell culture Ca⁺⁺ release assay) and may,therefore, constitute the optimal sequence and the optimal combinationof sequence elements

, B2, and

.

Additionally, 29 other MCP-1 binding nucleic acids were identified whichcannot be described by a combination of nucleotide sequence elements ashas been shown for Types 1-4 of MCP-1 binding nucleic acids. Thesesequences are listed in FIG. 7.

It is to be understood that any of the sequences shown in FIGS. 1through 7 are nucleic acids according to the present invention,including those truncated forms thereof but also including thoseextended forms thereof under the proviso, however, that the thustruncated and extended, respectively, nucleic acid molecules are stillcapable of binding to the target.

EXAMPLE 2 Nucleic Acids that Bind Murine MCP-1

Using biotinylated murine D-MCP-1 as a target, several nucleic acidmolecules binding thereto could be generated. The result of a sequenceanalysis of these nucleic acid molecules can be taken from FIG. 8.

The nucleic acids were characterized on the aptamer level using apull-down assay using biotinylated murine D-MCP-1 in order to in orderto rank them with respect to their binding behavior (Example 4).Selected sequences were synthesized as Spiegelmer (Example 3) and weretested using the natural configuration of MCP-1 (L-MCP) in an in vitrocell culture Ca⁺⁺-release (Example 5) and chemotaxis assay (Example 6).

As depicted in FIG. 8, D-188-A3-001 and D-189-G7-001 and theirderivatives bind D-MCP-1 with subnanomolar K_(D) in the pull-down assay(FIG. 8).

For D-mNOX-E36 (=D-188-A3-007; SEQ. ID No. 244), a dissociation constant(K_(D)) of 0.1-0.2 nM at 37° C. was determined (Example 4; FIG. 10). Therespective Spiegelmer mNOX-E36 (188-A3-007; SEQ. ID No. 122) exhibitedan inhibitory concentration (IC₅₀) of approx. 12 nM in an in vitroCa⁺⁺-release assay (Example 5; FIG. 13) and of approx. 7 nM in an invitro chemotaxis assay (Example 6; FIG. 16). For the PEGylatedderivative of mNOX-E36, mNOX-E36-3′PEG (SEQ. ID No. 254), IC₅₀'s ofapprox. 8 nM were determined in the Ca⁺⁺-release assay (Example 5, FIG.29) and approx. 3 nM in the chemotaxis assay (Example 6; FIG. 31).

It is to be understood that any of the sequences shown in FIGS. 1through 7 are nucleic acids according to the present invention,including those truncated forms thereof but also including thoseextended forms thereof under the proviso, however, that the thustruncated and extended, respectively, nucleic acid molecules are stillcapable of binding to the target.

EXAMPLE 3 Synthesis and Derivatization of Aptamers and Spiegelmers

Small Scale Synthesis

Aptamers and Spiegelmers were produced by solid-phase synthesis with anABI 394 synthesizer (Applied Biosystems. Foster City, Calif., USA) using2′TBDMS RNA phosphoramidite chemistry (M. J. Damha, K. K. Ogilvie,Methods in Molecular Biology, Vol. 20 Protocols for oligonucleotides andanalogs, ed. S. Agrawal, p. 81-114, Humana Press Inc. 1993). rA(N-Bz)-,rC(Ac)-, rG(N-ibu)-, and rU-phosphoramidites in the D- andL-configuration were purchased from ChemGenes, Wilmington, Mass.Aptamers and Spiegelmers were purified by gel electrophoresis.

Large Scale Synthesis Plus Modification

Spiegelmer NOX-E36 was produced by solid-phase synthesis with anÄktaPilot100 synthesizer (Amersham Biosciences; General ElectricHealthcare, Freiburg) using 2′TBDMS RNA phosphoramidite chemistry (M. J.Damha, K. K. Ogilvie, Methods in Molecular Biology, Vol. 20 Protocolsfor oligonucleotides and analogs, ed. S. Agrawal, p. 81-114, HumanaPress Inc. 1993). L-rA(N-Bz)-, L-rC(Ac)-, L-rG(N-ibu)-, andL-rU-phosphoramidites were purchased from ChemGenes, Wilmington, Mass.The 5′-amino-modifier was purchased from American InternationalChemicals Inc. (Framingham, Mass., USA). Synthesis of the unmodifiedSpiegelmer was started on L-riboG modified CPG pore size 1000 Å (LinkTechnology, Glasgow, UK), for the 3′-NH-modified Spiegelmer,3′-Aminomodifier-CPG, 1000 Å (ChemGenes, Wilmington, Mass.) was used.For coupling (15 min per cycle), 0.3 M benzylthiotetrazole(CMS-Chemicals, Abingdon, UK) in acetonitrile, and 3.5 equivalents ofthe respective 0.1 M phosphoramidite solution in acetonitrile was used.An oxidation-capping cycle was used. Further standard solvents andreagents for oligonucleotide synthesis were purchased from Biosolve(Valkenswaard, NL). The Spiegelmer was synthesized DMT-ON; afterdeprotection, it was purified via preparative RP-HPLC (Wincott F. et al.(1995) Nucleic Acids Res 23:2677) using Source15RPC medium (Amersham).The 5′DMT-group was removed with 80% acetic acid (30 min at RT).Subsequently, aqueous 2 M NaOAc solution was added and the Spiegelmerwas desalted by tangential-flow filtration using a 5 K regeneratedcellulose membrane (Millipore, Bedford, Mass.).

PEGylation of NOX-E36

In order to prolong the Spiegelmer's plasma residence time in vivo,Spiegelmer NOX-E36 was covalently coupled to a 40 kDa polyethyleneglycol (PEG) moiety at the 3′-end or 5′-end.

3′-PEGylation of NOX-E36

For PEGylation (for technical details of the method for PEGylation seeEuropean patent application EP 1 306 382), the purified 3′-aminomodified Spiegelmer was dissolved in a mixture of H₂O (2.5 ml), DMF (5ml), and buffer A (5 ml; prepared by mixing citric acid.H₂O [7 g], boricacid [3.54 g], phosphoric acid [2.26 ml], and 1 M NaOH [343 ml] andadding H2O to a final volume of 1 l; pH=8.4 was adjusted with 1 M HCl).

The pH of the Spiegelmer solution was brought to 8.4 with 1 M NaOH.Then, 40 kDa PEG-NHS ester (Nektar Therapeutics, Huntsville, Ala.) wasadded at 37° C. every 30 min in four portions of 0.6 equivalents until amaximal yield of 75 to 85% was reached. The pH of the reaction mixturewas kept at 8-8.5 with 1 M NaOH during addition of the PEG-NHS ester.

The reaction mixture was blended with 4 ml urea solution (8 M), 4 mlbuffer A, and 4 ml buffer B (0.1 M triethylammonium acetate in H₂O) andheated to 95° C. for 15 min. The PEGylated Spiegelmer was then purifiedby RP-HPLC with Source 15RPC medium (Amersham), using an acetonitrilegradient (buffer B; buffer C: 0.1 M triethylammonium acetate inacetonitrile). Excess PEG eluted at 5% buffer C, PEGylated Spiegelmer at10-15% buffer C. Product fractions with a purity of >95% (as assessed byHPLC) were combined and mixed with 40 ml 3 M NaOAC. The PEGylatedSpiegelmer was desalted by tangential-flow filtration (5 K regeneratedcellulose membrane, Millipore, Bedford Mass.).

5′-PEGylation of NOX-E36

For PEGylation (for technical details of the method for PEGylation seeEuropean patent application EP 1 306 382), the purified 5′-aminomodified Spiegelmer was dissolved in a mixture of H₂O (2.5 ml), DMF (5ml), and buffer A (5 ml; prepared by mixing citric acid.H₂O [7 g], boricacid [3.54 g], phosphoric acid [2.26 ml], and 1 M NaOH 1343 ml) andadding water to a final volume of 1 l; pH=8.4 was adjusted with 1 MHCl).

The pH of the Spiegelmer solution was brought to 8.4 with 1 M NaOH.Then, 40 kDa PEG-NHS ester (Nektar Therapeutics, Huntsville, Ala.) wasadded at 37° C. every 30 min in six portions of 0.25 equivalents until amaximal yield of 75 to 85% was reached. The pH of the reaction mixturewas kept at 8-8.5 with 1 M NaOH during addition of the PEG-NHS ester.

The reaction mixture was blended with 4 ml urea solution (8 M), and 4 mlbuffer B (0.1 M triethylammonium acetate in H₂O) and heated to 95° C.for 15 min. The PEGylated Spiegelmer was then purified by RP-HPLC withSource 15RPC medium (Amersham), using an acetonitrile gradient (bufferB; buffer C: 0.1 M triethylammonium acetate in acetonitrile). Excess PEGeluted at 5% buffer C, PEGylated Spiegelmer at 10-15% buffer C. Productfractions with a purity of >95% (as assessed by HPLC) were combined andmixed with 40 ml 3 M NaOAC. The PEGylated Spiegelmer was desalted bytangential-flow filtration (5 K regenerated cellulose membrane,Millipore, Bedford Mass.).

EXAMPLE 4 Determination of Binding Constants (Pull-Down Assay)

Direct Pull-Down Assay

The affinity of aptamers to D-MCP-1 was measured in a pull down assayformat at 20 or 37°C, respectively. Aptamers were 5′-phosphate labeledby T4 polynucleotide kinase (Invitrogen, Karlsruhe, Germany) using[γ-³²P]-labeled ATP (Hartmann Analytic, Braunschweig, Germany). Thespecific radioactivity of labeled aptamers was 200,000-800,000 cpm/pmol.Aptamers were incubated after de- and renaturation at 20 pMconcentration at 37° C. in selection buffer (20 mM Tris-HCl pH 7.4; 137mM NaCl; 5 mM KCl; 1 mM MgCl₂; 1 mM CaCl₂; 0.1% [w/vol] Tween-20)together with varying amounts of biotinylated D-MCP-1 for 4-12 hours inorder to reach equilibrium at low concentrations. Selection buffer wassupplemented with 10 μg/ml human serum albumin (Sigma-Aldrich,Steinheim, Germany), and 10 μg/ml yeast RNA (Ambion, Austin, USA) inorder to prevent adsorption of binding partners with surfaces of usedplasticware or the immobilization matrix. The concentration range ofbiotinylated D-MCP-1 was set from 8 pM to 100 nM; total reaction volumewas 1 ml. Peptide and peptide-aptamer complexes were immobilized on 1.5μl Streptavidin Ultralink Plus particles (Pierce Biotechnology,Rockford, USA) which had been preequilibrated with selection buffer andresuspended in a total volume of 6 μl. Particles were kept in suspensionfor 30 min at the respective temperature in a thermomixer. Immobilizedradioactivity was quantitated in a scintillation counter after detachingthe supernatant and appropriate washing. The percentage of binding wasplotted against the concentration of biotinylated D-MCP-1 anddissociation constants were obtained by using software algorithms(GRAFIT; Erithacus Software; Surrey U.K.) assuming a 1:1 stoichiometry.

Competitive Pull-Down Assay

In order to compare different D-MCP-1 binding aptamers, a competitiveranking assay was performed. For this purpose the most affine aptameravailable was radioactively labeled (see above) and served as reference.After de- and renaturation it was incubated at 37° C. with biotinylatedD-MCP-1 in 1 ml selection buffer at conditions that resulted in around5-10% binding to the peptide after immobilization and washing onNeutrAvidin agarose or Streptavidin Ultralink Plus (both from Pierce)without competition. An excess of de- and renatured non-labeled D-RNAaptamer variants was added to different concentrations (e.g. 2, 10, and50 nM) with the labeled reference aptamer to parallel binding reactions.The aptamers to be tested competed with the reference aptamer for targetbinding, thus decreasing the binding signal in dependence of theirbinding characteristics. The aptamer that was found most active in thisassay could then serve as a new reference for comparative analysis offurther aptamer variants.

EXAMPLE 5 Determination of Inhibitory Concentration in a Ca⁺⁺-ReleaseAssay

THP-1-cells (DSMZ, Braunschweig) were cultivated overnight at a celldensity of 0.3×10⁶/ml at 37° C. and 5% CO₂ in RPMI 1640 medium withGlutaMAX (Invitrogen) which contained in addition 10% fetal calf serum,50 units/ml penicillin, 50 μg/ml streptomycin and 50 μMβ-mercaptoethanol.

The Spiegelmers were incubated together with recombinant human MCP-1(Bachem) in Hanks balanced salt solution (HBSS), containing 1 mg/mlbovine serum albumin, 5 mM probenecid and 20 mM HEPES (HBSS+) for 15 to60 min at 37° C. in a 0.2 ml low profile 96-tube plate (“stimulationsolution”).

For loading with the calcium indicator dye, cells were centrifuged at300×g for 5 min, resuspended in 4 ml indicator dye solution (10 μMfluo-4 [Molecular Probes], 0.08% pluronic 127 [Molecular Probes] inHBSS+) and incubated for 60 min at 37° C. Thereafter, 11 ml HBSS+ wereadded and the cells were centrifuged as above, washed once with 15 mlHBSS+ and then resuspended in HBSS+ to give a cell density of1.1×10⁶/ml. 90 μl of this cell suspension were added to each well of ablack 96-well plate.

Measurement of fluorescence signals was done at an excitation wavelengthof 485 nm and an emission wavelength of 520 nm in a Fluostar Optimamultidetection plate reader (BMG). For parallel measurement of severalsamples, wells of one (perpendicular) row of a 96-well plate wererecorded together. First three readings with a time lag of 4 sec weredone for determination of the base line. Then the recording wasinterrupted and the plate was moved from the instrument. Using amulti-channel pipette, 10 μl of the stimulation solution was added tothe wells, then the plate was moved into the instrument again and themeasurement was continued. In total, 20 recordings with time intervalsof 4 seconds were performed.

For each well the difference between maximal fluorescence and base linevalue was determined and plotted against MCP-1 concentration or, in theexperiments on the inhibition of calcium release by Spiegelmers, againstconcentration of Spiegelmer.

Determination of Half-Maximal Effective Concentration (EC₅₀) for HumanMCP-1

After stimulation of THP-1 cells with various hMCP-1 concentrations andplotting the difference between the maximal and the baseline signals, adose-response curve for human MCP-1 was obtained, indicating a halfeffective concentration (EC₅₀) of about 2-4 nM (FIG. 11). Thisconcentration was used for the further experiments on inhibition ofCa⁺⁺-release by Spiegelmers.

Determination of Half-Maximal Effective Concentration (EC₅₀) for MarineMCP-11

After stimulation of THP-1 cells with various mMCP-1 concentrations andplotting the difference between the maximal and the baseline signals, adose-response curve for murine MCP-1 was obtained, indicating a halfeffective concentration (EC₅₀) of about 5 nM (FIG. 28).

This concentration was used for the further experiments on inhibition ofCa⁺⁺-release by Spiegelmers.

EXAMPLE 6 Determination of Inhibitory Concentration in a ChemotaxisAssay

THP-1 cells grown as described above were centrifuged, washed once inHBH (HBSS, containing 1 mg/ml bovine serum albumin and 20 mM HEPES) andresuspended at 3×10⁶ cells/ml. 100 μl of this suspension were added toTranswell inserts with 5 μm pores (Corning, #3421). In the lowercompartments MCP-1 was preincubated together with Spiegelmers in variousconcentrations in 600 μl HBH at 37° C. for 20 to 30 min prior toaddition of cells. Cells were allowed to migrate at 37° C. for 3 hours.Thereafter the inserts were removed and 60 μl of 440 μM resazurin(Sigma) in phosphate buffered saline was added to the lowercompartments. After incubation at 37° C. for 2.5 hours, fluorescence wasmeasured at an excitation wavelength of 544 nm and an emissionwavelength of 590 nm in a Fluostar Optima multidetection plate reader(BMG).

Determination of Half-Maximal Effective Concentration (EC₅₀) for HumanMCP-1

After 3 hours migration of THP-1 cells towards various human MCP-1concentrations, a dose-response curve for human MCP-1 was obtained,indicating a maximal effective concentration of about 1 nM and reducedactivation at higher concentrations (FIG. 14). For the furtherexperiments on inhibition of chemotaxis by Spiegelmers a MCP-1concentration of 0.5 nM was used.

Determination of Half-Maximal Effective Concentration (EC₅₀) for MarineMCP-1

After 3 hours migration of THP-1 cells towards various murine MCP-1concentrations, a dose-response curve for murine MCP-1 was obtained,indicating a maximal effective concentration of about 1-3 nM and reducedactivation at higher concentrations (FIG. 30). For the furtherexperiments on inhibition of chemotaxis by Spiegelmers a murine MCP-1concentration of 0.5 nM was used.

EXAMPLE 7 Binding Analysis by Surface Plasmon Resonance Measurement

7.1 Specificity Assessment of NOX-E36, 181-A2-018 and mNOX-E36

The Biacore 2000 instrument (Biacore AB, Uppsala, Sweden) was used toanalyze binding of nucleic acids to human MCP-1 and related proteins.When coupling was to be achieved via amine groups, the proteins weredialyzed against water for 1-2 h (Millipore VSWP mixed cellulose esters;pore size, 0.025 μM) to remove interfering amines. PioneerF1 or CM4sensor chips (Biacore AB) were activated before protein coupling by a35-μl injection of a 1:1 dilution of 0.4 M NHS and 0.1 M EDC at a flowof 5 μl/min. Chemokine was then injected in concentrations of 0.1-1.5μg/ml at a flow of 2 μl/min until the instrument's response was in therange of 1000-2000 RU (relative units). Unreacted NHS esters weredeactivated by injection of 35 μl ethanolamine hydrochloride solution(pH 8.5) at a flow of 5 μl/min. The sensor chip was primed twice withbinding buffer and equilibrated at 10 μl/min for 1-2 hours until thebaseline appeared stable. For all proteins, kinetic parameters anddissociation constants were evaluated by a series of Spiegelmerinjections at concentrations of 1000, 500, 250, 125, 62.5, 31.25, and 0nM in selection buffer (Tris-HCl, 20 mM; NaCl, 137 mM; KCl, 5 mM; CaCl₂,1 mM; MgCl₂, 1 mM; Tween20, 0.1% [w/v]; pH 7.4). In all experiments, theanalysis was performed at 37° C. using the Kinject command defining anassociation time of 180 and a dissociation time of 360 seconds at a flowof 10 μl/min. Data analysis and calculation of dissociation constants(K_(D)) was done with the BIAevaluation 3.0 software (BIACORE AB,Uppsala, Sweden) using the Langmuir 1:1 stochiometric fitting algorithm.

7.1.1 NOX-E36 and 181-A2-018 (Human-MCP-1 Specific Nucleic Acids)

Only for human MCP-1 all sensorgrams are depicted (FIGS. 17 and 20,respectively); for the other proteins, only the sensorgram obtained with125 nM Spiegelmer concentration is shown for sake of clarity (FIGS.18/19 and 21/22).

Analysis of the NOX-E36•hMCP-1 interaction: recombinant human MCP-1 wasimmobilized on a PioneerF1 sensor chip following the manufacturer'srecommendations (amine coupling procedure) until an instrument responseof 1381 RU (relative units) was established. The determined dissociationconstant (K_(D)) for NOX-E36 binding to human MCP-1 was ca. 890 pM (FIG.17).

Analysis of the 181-A2-018•hMCP-1 interaction: recombinant human MCP-1was immobilized on a CM4 sensor chip following the manufacturer'srecommendations (amine coupling procedure) until an instrument responseof 3111 RU (relative units) was established. The determined dissociationconstant (K_(D)) for 181-A2-018 binding to human MCP-1 was ca. 370 pM(FIG. 20).

To determine the specificity of NOX-E36 and 181-A2-018, various humanMCP-1 family proteins as well as human eotaxin were immobilized on aPioneerF1 and a CM4 sensor chip (hMCP-1, 1754 RU; hMCP-2, 1558 RU;hMCP-3, 1290 RU; eotaxin, 1523 RU). Kinetic analysis revealed thatNOX-E36 binds to eotaxin and hMCP-2 with dissociation constants (K_(D))of 5-10 nM; hMCP-3 was not recognized (FIGS. 18 and 24A). 181-A2-018, incontrast, binds eotaxin, hMCP-2 and hMCP-3, but with slightly loweraffinity (10-20 nM; FIGS. 21 and 24A).

Interspecies cross-reactivity of NOX-E36 and 181-A2-018 was assessedusing amino-coupling immobilized MCP-1 from human (1460 RU), monkey(1218 RU), pig (1428 RU), dog (1224 RU), rabbit (1244 RU), rat (1267RU), and mouse (1361 RU) on a PioneerF1 and a CM4 sensor chip. Kineticanalysis revealed that NOX-E36 binds to human, monkey, porcine, andcanine MCP-1 with comparable dissociation constants (K_(D)) of 0.89-1.2nM whereas MCP-1 from mouse, rat and rabbit were not recognized (FIGS.19 and 24A). 181-A2-018 binds to human and monkey MCP-1 with comparabledissociation constants (K_(D)) of 0.5-0.6 nM, whereas porcine, rabbitand canine MCP-1 are bound with much lower affinity. Rat and mouse MCP-1were not recognized by NOX-A2-018 (FIGS. 22 and 24A).

Sequences as well as degree of homology in percent identical amino acidsbetween the MCP-1 protein from different species and closely relatedhuman proteins are depicted in FIG. 23; calculated KD values for NOX-E36and 181-A2-018 are displayed in tabular format in FIG. 24A.

7.1.2 mNOX-E36 (Murine MCP-1 Specific Nucleic Acid)

To analyze the binding behaviour of mNOX-E36, 3759 RU of syntheticbiotinylated murine D-MCP-1 (flow cell 3) and 3326 RU of biotinylatedhuman D-MCP-1 (flow cell 4) were immobilized on a Streptavidinconjugated sensor chip (Biacore AB, Freiburg, Germany), respectively.mNOX-E36 aptamer (D-RNA) solutions of 500, 250, 125, 62.5, 31.25, and 0nM were injected using the Kinject command defining an association timeof 180 sec and a dissociation time of 360 sec. Flow cell 1 was used asbuffer and dextran matrix control (Biacore SA-Chip surface) whereas onflow cell 2, an unspecific D-peptide was immobilized to determineunspecific binding of the aptamer. FIG. 32 shows a sensorgram of theD-NOX-E36 kinetic for binding to murine D-MCP-1 with a calculateddissociation constant (K_(D)) of 200-300 pM. mNOX-E36 does not bindhuman D-MCP-1 (FIG. 33); for sake of clarity, only the sensorgramobtained with 125 nM Spiegelmer is shown.

7.2 Selectivity Assessment of NOX-E36

Selectivity of NOX-E36 was assessed by surface plasmon resonanceanalysis by immobilizing 5′biotinylated NOX-E36 on a Streptavidin(SA-Chip). 352 RU of NOX-E36 on flowcell (FC) 1 and equal amount of5′-terminal biotinylated non-functional control Spiegelmer (POC) on FC 2were immobilized by streptavidin/biotin binding. FC3 was used as surfacecontrol to determine unspecific binding to the dextran-SA sensorsurface.

100 nM of a panel of human chemokines from all four subgroups (CC, CXC,CX₃C, and XC) were injected for 360s and complexes were allowed todissociate for 360s at a flow of 10 μl/imin and 37° C. Response unitsafter association (Resp. 1; degree of interaction) and afterdissociation (Resp. 2, affinity of interaction) were plotted. After eachinjection the chip surface was regenerated with a 240s of 1 M sodiumchloride with 0.1% Tween; immobilized Spiegelmers were subsequentlyallowed to refold for 2 minutes at physiological conditions (runningbuffer). Injection of each chemokine was repeated 3 times. CXCL1, CXCL2,CXCL6 and CXCL9 showed unspecific binding to ribonucleic acids and chipdextran surface. Specific high-affinity binding to immobilized NOX-E36could only be detected for CCL2/MCP-1, CCL8/MCP-2, CCL11/eotaxin,CCL3/MIP1α, and CXCL7/NAP-2 (FIG. 24B). The finding that MCP-2 andcotaxin are bound by NOX-E36 is not surprising due to the relativelyhigh homology between these chemokines and MCP-1 of 62 and 70%, for theunexpected positives CCL3/MIP-1α and CXCL7/NAP-2, in vitro tests forfunctional inhibition have been performed or are currently beingestablished, respectively.

Finally, the kinetic parameters of interaction between NOX-E36 andCCL2/MCP-1, CCL8/MCP-2, CCL11/eotaxin, CCL3/MIP1α, CXCL7/NAP-2,CCL7/MCP-3 and CCL13/MCP-4 were determined in the “inverted” system.Here, the chemokines were immobilized and free NOX-E36 was injected (forthe detailed protocol, see 7.1). Kinetic data are summarized in FIG.24C.

7.3 Assessment of Anti-MIP-1α Functionality In Vitro

Biacore measurements had shown cross reactivity of NOX-E36 with MIP-1α.By employing a functional, cell culture-based in vitro assay it shouldbe checked if mere Biacore binding of NOX-E36 to MIP-1α also translatesto functionality, e.g. antagonism.

To achieve this, chemotaxis experiments with THP-1 cells were performedthat can be stimulated by MIP-1α. THP-1 cells grown as described abovewere centrifuged, washed once in HBH (HBSS, containing 1 mg ml bovineserum albumin and 20 mM HEPES) and resuspended at 3×10⁶ cells/ml. 100 μlof this suspension were added to Transwell inserts with 5 μm pores(Corning, #3421). In the lower compartments MIP-1α was preincubatedtogether with Spiegelmers in various concentrations in 600 μl HBH at 37°C. for 20 to 30 min prior to addition of cells. Cells were allowed tomigrate at 37° C. for 3 hours. Thereafter the inserts were removed and60 μl of 440 μM resazurin (Sigma) in phosphate buffered saline was addedto the lower compartments. After incubation at 37° C. for 2.5 hours,fluorescence was measured at an excitation wavelength of 544 nm and anemission wavelength of 590 nm in a Fluostar Optima multidetection platereader (BMG).

After 3 hours migration of THP-1 cells towards various human MIP-1αconcentrations, a dose-response curve for human MIP-1α was obtained,indicating a half-maximal effective concentration of about 1 nM andreduced activation at higher concentrations (FIG. 24D). For the furtherexperiments on inhibition of chemotaxis by Spiegelmers a MIP-1αconcentration of 0.5 nM was used.

Experiments for determination of chemotaxis inhibition by NOX-E36 wereperformed with a stimulus of 0.5 nM MIP-1α. It could be clearly shownthat NOX-E36 does not inhibit MIP-1α induced chemotaxis up to thehighest tested concentration of 1 μM MIP-1α. As positive control, therespective experiment with MCP-1 as stimulus was performed in parallel(FIG. 24E).

EXAMPLE 8 Therapy of Lupus-like Disease in MRL^(lpr/lpr) Mice withAnti-mMCP-1 Spiegelmer

Blocking proinflammatory mediators has become a successful approach forthe treatment of chronic inflammation (Steinman 2004). In addition toTNF and interleukins, CC-chemokines are important candidates forspecific antagonism because CC-chemokines mediate leukocyte recruitmentfrom the intravascular space to sites of inflammation (Baggiolini 1998,Luster 2005). There is very strong evidence that MCP-1 (=CCL2) and itsrespective chemokine receptor CCR2 play a crucial role in autoimmunetissue injury such as the clinical manifestations of systemic lupuserythematosus (Gerard & Rollins 2001). For example, MRL^(lpr/lpr) micedeficient either for the Ccl2 or the Ccr2 gene are protected fromlupus-like autoimmunity (Perez de Lema 2005, Tesch 1999). Hence, theCCL2/CCR2 axis may represent a promising therapeutic target, e.g. forlupus nephritis. In fact, delayed gene therapy or transfer oftransfected cells, both resulting in in situ production of anNH₂-truncated MCP-1, markedly reduced autoimmune tissue injury inMRL^(lpr/lpr) mice. However, such experimental approaches cannot be usedin humans because of irrepressible antagonist production and tumorformation (Hasegawa 2003, Shimizu 2004). Therefore, it remains necessaryto develop novel CCL2 antagonists with favorable pharmacokineticprofiles in vivo. In this example it is shown that blockade of murineCCL2 with the anti-mCCL2 Spiegelmer mNOX-E36 or mNOX-E36-3′PEG would besuitable for the treatment of lupus nephritis and other diseasemanifestations of systemic lupus erythematosus. Late onset of mCCL2Spiegelmer therapy effectively improves lupus nephritis, autoimmuneperibronchitis, and lupus-like skin disease in MRL^(lpr/lpr) mice,independent of any previous problem associated with therapeuticCCL2/CCR2 blockade.

Animals and Experimental Protocol

Ten week old female MRL^(lpr/lpr) mice were obtained from HarlanWinkelmann (Borchen, Germany) and kept under normal housing conditionsin a 12 hour light and dark cycle. Water and standard chow (Ssniff,Soest, Germany) were available ad libitum. At age 14 weeks, groups of 12mice received subcutaneous injections of Spiegelmers in 5% glucose(injection volume, 4 ml/kg) three times per week as follows: mNOX-E36,1.5 μmol/kg; mNOX-E36-3′PEG, 0.9 μmol/kg; nonfunctional controlSpiegelmer PoC (5′-UAAGGAAACUCGGUCUGAUGCGGU AGCGCUGUGCAGAGCU-3′), 1.9μmol/kg; PoC-PEG, 0.9 μmol/kg; vehicle (5% glucose). The plasma levelsof mNOX-E36 and mNOX-E36-3′PEG were determined from blood samples takenweekly from the retroorbital sinus 3 or 24 hours after injection,respectively. Spiegelmer levels in plasma samples were determined by amodification of the sandwich hybridization method as described inExample 8. Mice were sacrificed by cervical dislocation at the end ofweek 24 of age.

Evaluation of Systemic Lupus

Skin lesions were recorded by a semiquantitative score (Schwarting2005). The weight ratio of spleen and the bulk of mesenterial lymphnodesto total body weight were calculated as markers of the lupus-associatedlymphoproliferative syndrome. Blood and urine samples were collectedfrom each animal at the end of the study period by bleeding from theretro-orbital venous plexus under general anesthesia with inhaled ether.Blood and urine samples were collected from each animal at the end ofthe study and urine albumin/creatinine ratio and serum dsDNAautoantibody IgG isotype titers were determined as previously described(Pawar 2006). Glomerular filtration rate (GFR) was determined at 24weeks by clearance kinetics of plasma FITC-inulin (Sigma-Aldrich,Steinheim. Germany) 5, 10, 15, 20, 35, 60, and 90 minutes after a singlebolus injection (Qi 2004). Fluorescence was determined with 485 nmexcitation and read at 535 nm emission. GFR was calculated based on atwo-compartment model using a non-linear regression curve-fittingsoftware (GraphPad Prism, GraphPad Software Inc., San Diego, Calif.).Serum cytokine levels were determined using commercial ELISA kits forIL-6, IL-12p40 (OptEiA, BD Pharmingen), and IFN-α (PBL Biomedical Labs,USA). From all mice, kidneys and lungs were fixed in 10% bufferedformalin, processed, and embedded in paraffin. 5-μm sections for silverand periodic acid-Schiff stains were prepared following routineprotocols (Anders 2002). The severity of the renal lesions was gradedusing the indices for activity and chronicity as described for humanlupus nephritis (Austin 1984), and morphometry of renal interstitialinjury was conducted as previously described (Anders 2002). The severityof the peribronchial inflammation was graded semiquantitatively from0-4. For immunostaining, sections of formalin-fixed andparaffin-embedded tissues were dewaxed and rehydrated. Endogenousperoxidase was blocked by 3% hydrogen peroxide and antigen retrieval wasperformed in Antigen Retrieval Solution (Vector, Burlingame, Calif.) inan autoclave oven. Biotin was blocked using the AvidiniBiotin blockingKit (Vector). Slides were incubated with the primary antibodies for onehour, followed by biotinylated secondary antibodies (anti-rat IgG,Vector), and the ABC reagent (Vector). Slides were washed in phosphatebuffered saline between the incubation steps. 3′3′Diaminobenzidine (DAB,Sigma, Taufkirchen, Germany) with metal enhancement was used asdetection system, resulting in a black colour product. Methyl green wasused as counterstain, slides were dehydrated and mounted in Histomount(Zymed Laboratories, San Francisco, Calif.).

The following primary antibodies were used: rat anti-Mac2 (macrophages,Cederlane, Ontario, Canada, 1:50), anti-mouse CD3 (1:100, clone 500A2,BD), anti-mouse IgG₁ (1:100, M32015, Caltag Laboratories, Burlingame,Calif., USA), anti-mouse IgG_(2a) (1:100, M32215, Caltag), anti-mouse C3(1:200, GAM/C3c/FITC, Nordic Immunological Laboratories, Tilburg,Netherlands). Negative controls included incubation with a respectiveisotype antibody. For quantitative analysis glomerular cells werecounted in 15 conical glomeruli per section. Glomerular Ig and C3cdeposits were scored from 0-3 on 15 conical glomerular sections.

RNA Preparation and Real-Time Quantitative (TaqMan) RT-PCR

Renal tissue from each mouse was snap frozen in liquid nitrogen andstored at −80° C. From each animal, total renal RNA preparation andreverse transcription were performed as described (Anders 2002). Primersand probes were from PE Biosystems, Weiterstadt, Germany. The usedprimers (300 nM) used for detection of Ccl2, Ccl5 and 18S rRNA,predeveloped TaqMan assay reagent from PE Biosystems.

Flow Cytometry

Total blood and bone marrow samples were obtained from mice of allgroups at the end of the study. Flow cytometry was performed using aFACScalibur machine and the previously characterized MC21 anti-mCCR2antibody (Mack 2001). A biotinylated anti-rat IgG antibody (BDBiosciences) was used for detection. A rat IgG_(2b) (BD Biosciences) wasused as isotype control.

Statistical Analysis

Data were expressed as mean±standard error of the mean (SEM). Comparisonbetween groups were performed using univariate ANOVA. PosthocBonferroni's correction was used for multiple comparisons. A value ofp<0.05 was considered to indicate statistical significance.

Sandwich Hybridisation Assay

Amount of Spiegelmer in the samples was quantified by a sandwichhybridisation assay based on an assay as described by Drolet et al. 2000(Pharm Res 17:1503). Blood samples were collected in parallel to followthe plasma clearance of NOX-E36. Selected tissues were prepared todetermine Spiegelmer concentrations.

Hybridisation Plate Reparation

Spiegelmer mNOX-E36 was quantified by using a non-validated sandwichhybridisation assay. Briefly, the mNOX-E36 capture probe (Seq. ID.: 281)was immobilized to white DNA-BIND 96 well plates (Corning Costar,Wiesbaden, Germany) at 0.75 mM in 0.5 M sodium phosphate, 1 mM EDTA, pH8.5 over night at 4° C. Wells were washed twice and blocked with 0.5%w/v BSA in 0.25 M sodium phosphate, 1 mM EDTA, pH 8.5 for 3 h at 37° C.,washed again and stored at 4° C. until use. Prior to hybridisation,wells were pre-warmed to 37° C. and washed twice with pre-warmed washbuffer (3×SSC, 0.5% [w/v] sodium dodecyl sarcosinate, pH 7.0; in advancea 20× stock [3 M NaCl, 0.3 M Na₃Citrate) is prepared without sodiumlauroylsarcosine and diluted accordingly).

Sample Preparation

All samples were assayed in duplicates. Plasma samples were thawed onice, vortexed and spun down briefly in a cooled tabletop centrifuge.Tissue homogenates were thawed at RT and centrifuged 5 min at maximumspeed and RT. Only 5 μl each sample were removed for the assay, andafterwards returned to the freezer for storage. Samples were dilutedwith hybridisation buffer (8 nM mNOX-E36 detection probe [Seq. ID:282]in wash buffer) at RT according to the following scheme:

1:30  5 μl sample + 145 μl hybridisation buffer 1:300 20 μl 1:30 + 180μl hybridisation buffer 1:3000 20 μl 1:300 + 180 μl hybridisation buffer1:30000 20 μl 1:3000 + 180 μl hybridisation buffer

All sample dilutions were assayed, mNOX-E36 standard was serial dilutedto a 8-point calibration curve spanning the 0-4 nM range. No QC sampleswere prepared and assayed. Calibration standard was identical to that ofthe in-study samples.

Hybridisation and Detection

Samples were heated for 10 min at 95° C. and cooled to 37° C.Spiegelmer/detection probe complexes were annealed to immobilizedcapture probes for 30 min at 37° C. Unbound spiegelmers were removed bywashing twice with wash buffer and 1×TBST (20 mM Tris-Cl, 137 mM NaCl,0.1% Tween 20, pH 7.5), respectively. Hybridized complexes were detectedby streptavidin alkaline phosphatase diluted 1:5000 in 1×TBST for 1 h atroom temperature. To remove unbound conjugate, wells were washed againwith 1×TBST and 20 mM Tris-Cl, 1 mM MgCl2, pH 9.8 (twice each). Wellswere finally filled with 100 ml CSDP substrate (Applied Biosystems,Darmstadt, Germany) and incubated for 45 min at room temperature.Chemiluminescence was measured on a FLUOstar Optima microplate reader(BMG Labtechnologies, Offenburg, Germany).

Data Analysis

The following assayed sample dilutions were used for quantitative dataanalysis:

rat EDTA plasma 1:2000

The data obtained from the vehicle group (no Spiegelmer was adminstered)was subtracted as background signal.

The sandwich hybridisation assay as described herein also works insimilar fashion for Spiegelmer NOX-36, NOX-E36-5′-PEG and NOX-E36-3′-PEGwhereby the respective NOX-E36 capture probe (Seq. ID:255) and therespective NOX-E36 detection probe (Seq. ID:256) has to be used (datanot shown).

Results

mNOX-E36-3′PEG Improves Survival and Kidney Disease of MRL^(lpr/lpr)Mice

Female MRL^(lpr/lpr) mice develop and subsequentially die fromproliferative immune complex glomerulonephritis with strikingsimilarities to diffuse proliferative lupus nephritis in humans. In thistherapeutic study design, treated MRL^(lpr/lpr) mice were treated withpegylated and unpegylated anti-mCCL2 Spiegelmer, pegylated andunpegylated control (“PoC”)-Spiegelmer or vehicle from week 14 to 24 ofage. At this time point vehicle, PoC or PoC-PEG-treated MRL^(lpr/lpr)mice showed diffuse proliferative glomerulonephritis characterized byglomerular macrophage infiltration and a mixed periglomerular andinterstitial inflammatory cell infiltrate consting of glomerular andinterstitial Mac2-positive macrophages and interstitial CD3-positivelymphocytes (FIGS. 34 and 35). mNOX-E36-3′PEG improved the activity andchronicity index of lupus nephritis as well as the forementioned markersof renal inflammation (FIG. 35). The unpegylated molecule mNOX-E36 wasless effective on the chronicity index and interstitial macrophage and Tcell counts (FIG. 35). Advanced chronic kidney disease was furtherillustrated by tubular atrophy and confluent areas of interstitialfibrosis in vehicle-, PoC-, and PoC-PEG-treated mice (FIG. 34). Applyingmorphometry to quantify these changes, it was found that pegylated andunpegylated mNOX-E36 reduced interstitial volume, tubular cell damage,and tubular dilation, all being markers of the severity and prognosis ofchronic kidney disease (FIG. 36). mNOX-E36-3′PEG but not unpegylatedmNOX-E36 improved 50% mortality (FIG. 37). Thus, mNOX-E36-3′PEG canreduce the number of renal macrophage and T cell infiltrates and improvelupus nephritis and (renal) survival of MRL^(lpr/lpr) mice. In order tostudy whether treatment with mNOX-E36 and mNOX-E36-3′PEG affectsintrarenal inflammation in MRL^(lpr/lpr) mice, real-time RT-PCR wasperformed to assess the expression levels of the proinflammatorychemokines CCL2 and CCL5 which were previously shown to be progressivelyupregulated in kidneys of MRL^(lpr/lpr) mice during progression of renaldisease (Perez de Lema 2001). Treatment with mNOX-E36 and mNOX-E36-3′PEGfrom week 14 to 24 of age reduced renal expression of CCL2 and CCL5 mRNAcompared to vehicle-treated controls (FIG. 38).

Anti-CCL2 Spiegelmers Reduce Extrarenal Autoimmune Tissue Injury inMRL^(lpr/lpr) Mice

Skin and lungs are also commonly affected from autoimmune tissue injuryin MRL^(lpr/lpr) mice. In vehicle-treated mice autoimmune lung diseasewas characterized by moderate peribronchiolar and perivascularinflammatory cell infiltrates and skin lesions were observed in 60% ofmice (FIGS. 39, 40 and 35). mNOX-E36 and mNOX-E36-3′PEG both reducedperibronchial inflammation and skin disease as compared to vehicle-,PoC-, and PoC-PEG-treated MRL^(lpr/lpr) mice, respectively (FIGS. 39, 40and 35). Hence, the effects of CCL2-specific Spiegelmers are not limitedto lupus nephritis but extend to other manifestations of autoimmunetissue injury in MRL^(lpr/lpr) mice.

mNOX-E36 and the Lymphoproliferative Syndrome, dsDNA Autoantibodies, andSerum Cytokine Levels in MRL^(lpr/lpr) Mice

Female MRL^(lpr/lpr) mice develop a lymphoproliferative syndromecharacterized by massive splenomegaly and bulks of cervical, axillary,inguinal, and mesenterial lymph nodes. mNOX-E36 and mNOX-E36-3′PEG bothhad no effect on the weight of spleens and lymph nodes in MRL^(lpr/lpr)mice (FIG. 41). Autoimmunity in MRL^(lpr/lpr) mice is characterized bythe production of autoantibodies against multiple nuclear antigensincluding dsDNA. In 24 week old MRL^(lpr/lpr) mice serum dsDNA IgG,IgG₁, IgG_(2a), IgG_(2b) autoantibodies were present at high levels.mNOX-E36 and mNOX-E36-3′PEG both had no effect on either of these DNAautoantibodies (FIG. 41). Lupus-like disease in vehicle-treatedMRL^(lpr/lpr) mice was characterized by elevated serum levels of IFN-α,IL-12p40, and IL-6. mNOX-E36 and mNOX-E36-3′PEG both had no effect oneither of these inflammatory mediators (FIG. 41). Thus, both mNOX-E36variants do not affect lymphoproliferation, anti-dsDNA IgG production,and serum cytokine levels in MRL^(lpr/lpr) mice.

Plasma Levels of mNOX-E36 and mNOX-E36-3′PEG in MRL^(lpr/lpr) Mice

mNOX-E36 and mNOX-E36-3′PEG plasma levels were determined at weeklyintervals in order to monitor drug exposure during progressive kidneydisease of MRL^(lpr/lpr) mice. The median plasma levels of mNOX-E36 3 hafter injection and mNOX-E36-3′PEG 24 h after injection wereapproximately 300 nM and 1 μM throughout the study, respectively (FIG.42). Thus, pegylation increased the plasma levels of mNOX-E36 and theprogressive kidney disease of MRL^(lpr/lpr) mice did not modulate thepharmacokinetics of both Spiegelmers.

mNOX-E36-3′PEG Blocks the Emigration of Monocytes from the Bone Marrow

Monocyte emigration from bone marrow during bacterial infection wasshown to involve chemokine receptor CCR2 (Serbina 2006), but the role ofCCL2 in the context of autoimmunity remains hypothetical. Therefore, theCCR2-positive monocyte population in peripheral blood and bone marrowsin mice of mNOX-E36-3′PEG- and vehicle-treated groups of 24 week oldMRL^(lpr/lpr) mice was examined. Treatment with mNOX-E36-3′PEG increasedthe percentage of CCR2 positive cells in the bone marrow from 13% to 26%whereas it reduced this population in the peripheral blood from 26% to11% (FIG. 43). These data support a role of CCL2 for the evasion of CCR2positive cells from the bone marrow during autoimmune disease ofMRL^(lpr/lpr) mice.

SUMMARY

Applying the Spiegelmer technology, a novel and specific mCCL2antagonist was created which potently blocks mCCL2 in vitro and in vivo.In fact, late onset of treatment with the CCL2 Spiegelmer markedlyimproved advanced lupus-like autoimmune tissue injury in MRL^(lpr/lpr)mice. These data support a central role for CCL2 in chronic inflammatorytissue damage and identify CCL2 Spiegelmers as a novel therapeutic forautoimmune tissue injury.

EXAMPLE 9 Therapy of Diabetic Nephropathy in Unilaterally NephrectomizedDiabetic Mice with Anti-mMCP-1 Spiegelmer

Diabetic nephropathy remains a leading cause of end-stage renal diseasebecause targeting the angiotensin-dependent pathomechanisms does notalways prevent disease progression (Zimmet 2001; Ritz 1999; UnitedStates Renal Data System 2004; Svensson 2003). Hence, other treatmentstrategies are required to add on to the therapeutic armament fordiabetic nephropathy.

Data from recent experimental studies relate the progression of diabeticnephropathy to intrarenal inflammation (Galkina 2006; Mora 2005; Meyer2003; Tuttle 2005). For example, mycophenolate mofetil, methotrexate orirradiation reduce urinary albumin excretion, and glomerulosclerosis inrats with streptozotocin-induced diabetic nephropathy (Yozai 2005;Utimura 2003). Yet, the molecular and cellular mechanisms of intrarenalinflammation in diabetic nephropathy remain poorly characterized.Patients with diabetic nephropathy have increased serum levels of acutephase markers of inflammation but this may not represent intrarenalinflammation (Dalla Vestra 2005; Navarro 2003). Patients with diabeticnephropathy excrete high levels of the CC-chemokine monocytechemoattractant protein 1 (MCP-1/CCL2) in the urine which may be morespecific for intrarenal inflammation (Morii 2003; Tashiro 2002;Takebayashi 2006). In fact, MCP-1/CCL2 is expressed by human mesangialcells exposed to either high glucose concentrations or advancedglycation end products (Ihm 1998; Yamagishi 2002). CCL2 is involved inthe complex multistep process of leukocyte recruitment fromintravascular to extravascular compartments, i.e. glomeruli and therenal interstitium (Baggiolini 1998). In fact, macrophage infiltratesare a common finding in human and experimental diabeticglomerulosclerosis and tubulointerstitial injury (Bohle 1991; Furuta1993; Chow 2007). Ccl2-deficient type 1 or type 2 diabetic mice havelower glomerular macrophage counts which is associated with lessglomerular injury (Chow 2004; Chow 2006). In these studies thefunctional role of CCL2 for glomerular pathology of type 1 and type 2diabetic nephropathy was also demonstrated. Hence, CCL2 may represent apotential therapeutic target for diabetic nephropathy, and suitable CCL2antagonists with favourable pharmacokinetic profiles should be validatedin this disease context. In this example we report the effects of thePEGylated anti-CCL2 Spiegelmer mNOX-E36-3′PEG in type 2 diabetic db/dbmice with advanced diabetic nephropathy. We shown that ananti-CCL2-Spiegelmer would be suitable for the treatment of diabeticnephropathy.

Animals and Experimental Protocol

Male 5 week old C57BLKS db/db or C57BLKS wild-type mice were obtainedfrom Taconic (Ry, Denmark) and housed in filter top cages with a 12 hourdark/light cycle and unlimited access to food and water for the durationof the study. Cages, bedding, nestlets, food, and water were sterilizedby autoclaving before use. At the age of 6 weeks uninephrectomy (“1K”mice) or sham surgery (“2K” mice) was performed through a 1 cm flankincision as previously described in db/db and wild-type mice (Bower1980). In mice of the sham surgery groups the kidney was left in situ.10 weeks later, at the age of 4 months, 1 K db/db mice were divided intwo groups that received three times per week subcutaneous injectionswith either mNOX-E36-3′PEG or PoC-PEG in 5% glucose (dose, 0.9 μmol/kg;injection volume, 1 ml/kg). Treatment was continued for 8 weeks (untilthe age 6 months) when the animals were sacrificed and the tissues wereobtained for histopathological evaluation. All experimental procedureshad been approved by the local government authorities.

Evaluation of Diabetic Nephropathy

All immunohistological studies were performed on paraffin-embeddedsections as described (Anders 2002). The following antibodies were usedas primary antibodies: rat anti-Mac2 (glomerular macrophages, Cederlane,Ontario, Canada, 1:50), anti-Ki-67 (cell proliferation, Dianova,Hamburg, Germany, 1:25). For histopathological evaluation, from eachmouse parts of the kidneys were fixed in 10% formalin inphosphate-buffered saline and embedded in paraffin. 3 μm-sections werestained with periodic acid-Schiff reagent or silver following theinstructions of the supplier (Bio-Optica, Milano, Italy). Glomerularsclerotic lesions were assessed using a semiquantitative score by ablinded observer as follows: 0=no lesion, 1=<25% sclerotic, 2=25-49%sclerotic, 3=50-74% sclerotic, 4=75-100% sclerotic, respectively. 15glomeruli were analysed per section. The indices for interstitial volumeand tubular dilatation were determined by superimposing a grid of 100points on 10 non-overlapping cortical fields as described previously(Anders 2002) Interstitial cell counts were determined in 15 high powerfields (hpf, 400×) by a blinded observer. RNA preparation and real-timequantitative (TaqMan) RT-PCR was done from deparaffinized glomeruli.After incubation in lysing buffer (10 mM Tris-HCl, 0.1 mM EDTA, 2% SDSand 20 μg/ml proteinase K) for 16 h at 60° C., phenol-chloroform-basedRNA extraction was performed. Glomerular RNA was dissolved in 10 μlRNAse free water. Reverse transcription and real time RT-PCR from totalorgan and glomerular RNA was performed as described (Anders 2002, Cohen2002). Controls consisting of ddH₂O were negative for target andhousekeeper genes. Oligonucleotide primer (300 nM) and probes (100 nM)for mCcl2, Gapdh, and 18 S rRNA were predeveloped TaqMan assay reagentsfrom PE. Primers and probes were from ABI Biosystems, Weiterstadt,Germany. Glomerular filtration rate (GFR) was determined by clearancekinetics of plasma FITC-inulin (Sigma-Aldrich, Steinheim, Germany) 5,10, 15, 20, 35, 60, and 90 minutes after a single bolus injection (Qi2004). Fluorescence was determined with 485 nm excitation and read at535 nm emission. GFR was calculated based on a two-compartment modelusing a non-linear regression curve-fitting software (GraphPad Prism,GraphPad Software Inc., San Diego, Calif.). All data are presented asmean±SEM. Comparison of groups was performed using ANOVA and post-hocBonferroni's correction was used for multiple comparisons. A value ofp<0.05 was considered to indicate statistical significance.

Results

mNOX-E36-3′PEG Reduces Glomerular Macrophage Counts and GlobalGlomerulosclerosis in Unilaterally Nephrectomized db/db Mice

When lack of functional CCL2 is associated with decreased glomerularmacrophage recruitment in db/db mice (Chow 2007) and mNOX-E36-3′PEG isable to block CCL2-mediated macrophage recruitment in vitro and in vivo,mNOX-E36-3′PEG should impair renal macrophage recruitment in db/db micewith advanced type 2 diabetic nephropathy. To test this hypothesis, weinitiated subcutaneous injections with mNOX-E36-3′PEG or PoC-PEG at ageof 4 months in unilaterally nephrectomized (“1K”) db/db mice. Treatmentwas continued for K weeks when tissues were collected for the assessmentof diabetic nephropathy. During that period, mNOX-E36-3′PEG treatmentdid not significantly affect white blood or platelet counts, bloodglucose levels or body weight which were both markedly elevated in allgroups of db/db mice as compared to non-diabetic BLKS mice (data notshown). Interestingly, mNOX-E36-3′PEG increased the serum levels of CCL2in 1K db/db mice, indicating that the CCL2 antagonist retains CCL2 inthe circulation (FIG. 44). Consistent with our hypothesis mNOX-E36-3′PEGsignificantly reduced the number of glomerular macrophages by 40% ascompared to PoC-PEG- or vehicle-treated db/db mice, associated withlower numbers of Ki-67 positive proliferating cells within theglomerulus in mNOX-E36-3′PEG-treated db/db mice (FIG. 45). Thesefindings were associated with a significant improvement of globaldiabetic glomerulosclerosis in 1K db/db mice (FIG. 46). In fact,mNOX-E36-3′PEG treatment reduced diabetic glomerulosclerosis in 1K db/dbmice to the extent of glomerulosclerosis present in age-matchednon-nephrectomized (“2K”) db/db mice (FIG. 46). These findings show thatdelayed blockade of CCL2-dependent glomerular macrophage recruitmentwith mNOX-E36-3′PEG prevents global diabetic glomerulosclerosis in type2 diabetic db/db mice.

mNOX-E36-3′PEG Improves GFR in 1K db/db Mice

The beneficial effects of mNOX-E36-3′PEG treatment on diabeticglomerulosclerosis in 1K db/db mice should be associated with a betterGFR. We analyzed FITC-inulin clearance kinetics as a marker of GFR indb/db mice (Qi 2004). As compared to a normal GFR of about 250 ml/min indb/db mice (Qi 2004), we found a reduced GFR of was 112±23 ml/min in 6months old 1K db/db mice injected with PoC-PEG (FIG. 47). mNOX-E36-3′PEGtreatment significantly improved the GFR to 231±30 ml/min in 1K db/dbmice (p<0.001) suggesting that blocking CCL2-dependent glomerularmacrophage recruitment can also improve renal function in type 2diabetic mice.

mNOX-E36-3′PEG Reduces Interstitial Macrophage Counts andTubulointerstitial Injury in 1K db/db Mice

Advanced diabetic nephropathy in humans is associated with significantnumbers of interstitial macrophages and tubulointerstitial injury (Bohle1991). In 2K db/db mice interstitial macrophage infiltrates andsignificant tubulointerstitial injury does not occur before 8 months ofage (Chow 2007). Early uninephrectomy accelerates the development oftubulointerstitial pathology in db/db mice (Ninichuk 2005), thus wequantified interstitial macrophages, tubular dilatation and interstitialvolume as markers of tubulointerstitial damage in mice of all groups at6 months of age. At this time point 1K db/db mice revealed increasednumbers of interstitial macrophages and significant elevations oftubular dilatation and interstitial volume as compared to 2K db/db mice(FIG. 45, FIG. 48). mNOX-E36-3′PEG treatment reduced the numbers ofinterstitial macrophages by 53% as well as tubular dilatation andinterstitial volume in 1K db/db mice (FIG. 45, FIG. 48). Thus, blockingCCL2-dependent renal macrophage recruitment also preventstubulointerstitial injury in type 2 diabetic db/db mice.

mNOX-E36-3′PEG Reduces Renal Expression of (C12 in 1K db/db Mice

Macrophage infiltrates amplify inflammatory responses in tissue injury,e.g. local CCL2 expression. We therefore hypothesized that themNOX-E36-3′PEG-related decrease in renal macrophages would be associatedwith less renal CCL2 expression. We used real-time RT-PCR to quantifythe mRNA expression of CCL2 in db/db mice. mNOX-E36-3′PEG reduced themRNA levels of CCL2 in kidneys of 6 months old 1 K db/db mice ascompared to age-matched PoC-PEG-treated mice (FIG. 49). To furtherassess the spatial expression of CCL2 we performed immunostaining forCCL2 protein on renal sections. In 1K db/db mice the expression of CCL2was markedly enhanced in glomeruli, tubuli, and interstitial cells ascompared to 2K db/db or 2K wild-type mice (FIG. 50). mNOX-E36-3′PEGmarkedly reduced the staining for CCL2 in all these compartments ascompared to vehicle- or PoC-PEG-treated 1K db/db mice. These dataindicate that blocking CCL2-dependent renal macrophage recruitment withmNOX-E36-3′PEG reduces the local expression of CCL2 in 1K db/db mice.

SUMMARY

The concept that inflammation contributies to the progression of humandiabetic nephropathy becomes increasingly accepted (Tuttle 2005),bringing MCP-1/CCL2 as a potential target to treat this disease into thefocus. In this example, we have shown that treatment of unilaterallynephrectomized diabetic mice with mNOX-E36-3′PEG reduced the numbers ofglomerular (and interstitial) macrophages at 6 months of age, associatedwith less proliferating glomerular cells. In addition, renal/glomerularexpression of CCL2 mRNA was markedly reduced with mNOX-E36-3′PEGtreatment. Furthermore, lower numbers of glomerular macrophages andglomerular proliferating cells in the therapy group were associated withprotection from global glomerulosclerosis and with a significantimprovement of the glomerular filtration rate. The beneficial effects ofmNOX-E36-3′PEG on glomerular pathology and renal function in diabeticmice are consistent with those studies that have used other CCL2antagonists in other models of glomerular injury (Lloyd 1997, Hasegawa2003, Tang 1996, Wenzel 1997, Fujinaka 1997, Schneider 1999).Remarkably. delayed onset of CCL2 blockade also reduced the numbers ofinterstitial macrophages being associated with less tubulointerstitialpathology in 1 K db/db mice.

Together, these data validate CCL2 as a promising therapeutic target fordiabetic nephropathy and suggest that initiating CCL2 blockade with aSpiegelmer—even at an advanced stage of the disease—may still beprotective.

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The features of the present invention disclosed in the specification,the claims and/or the drawings may both separately and in anycombination thereof be material for realizing the invention in variousforms thereof.

The invention claimed is:
 1. An L-nucleic acid that binds MCP-1,comprising in 5′→3′ direction, a first stretch Box B1A, a second stretchBox B2A, a third stretch Box B3, a fourth stretch Box B2B, a fifthstretch Box B4, a sixth stretch Box B5A, a seventh stretch Box B6, aneighth stretch Box B5B and a ninth stretch Box B1B, wherein a. the firststretch Box B1A and the ninth stretch Box B1B optionally hybridize witheach other to form a double-stranded structure; b. the second strand BoxB2A and the fourth Box B2B optionally hybridize with each other to forma double-stranded structure; c. the sixth stretch Box B5A and the eighthBox B5B optionally hybridize with each other to form a double-strandedstructure; d. the first stretch Box B1A comprises GURCUGC, GKSYGC, KBBSCor BNGC; e. the second stretch Box B2A comprises GKMGU; f. the thirdstretch Box B3 comprises KRRAR; g. the fourth stretch Box B2B comprisesACKMC; h. the fifth stretch Box B4 comprises CURYGA, CUWAUGA, CWRMGACWor UGCCAGUG; i. the sixth stretch Box B5A comprises GGY or CWGC; j. theseventh stretch Box B6 comprises YAGA, CKAAU or CCUUUAU; k. the eighthstretch Box B5B comprises GCYR or GCWG; and l. the ninth stretch Box B1Bcomprises GCAGCAC, GCRSMC, GSVVM or GCNV.
 2. The nucleic acid accordingto claim 1, wherein the third stretch Box B3 comprises GAGAA or UAAAA.3. The nucleic acid according to claim 1, wherein the fifth stretch BoxB4 comprises CAGCGACU or CAACGACU.
 4. The nucleic acid according toclaim 1, wherein the seventh stretch Box B6 comprises UAGA.
 5. Thenucleic acid according to claim 1, wherein a) the first stretch Box B1Acomprises GURCUGC and the ninth stretch Box B1B comprises GCAGCAC; b)the first stretch Box B1A comprises GKSYGC and the ninth stretch Box B1Bcomprises GCRSMC; c) the first stretch Box B1A comprises KBBSC and theninth stretch Box B1B comprises GSVVM; or d) the first stretch Box B1Acomprises BNGC and the ninth stretch Box B1B comprises GCNV.
 6. Thenucleic acid according to claim 1, wherein the second stretch Box B2Acomprises GKMGU and the fourth stretch Box B2B comprises ACKMC.
 7. Thenucleic acid according to claim 1, wherein a) the sixth stretch Box B5Acomprises GGY and the eighth stretch Box B5B comprises GCYR; or b) thesixth stretch Box B5A comprises CWGC and the eighth stretch Box B5Bcomprises GCWG.
 8. The nucleic acid according to claim 1, wherein thenucleic acid comprises one of SEQ ID NOs:56 to 61, SEQ ID NOs:67 to 71or SEQ ID NO:73.
 9. The nucleic acid according to claim 1, wherein thenucleic acid binds eotaxin, MCP-1, MCP-2 or MCP-3.
 10. The nucleic acidaccording to claim 1, wherein the nucleic acid comprises a highmolecular weight moiety or a modification increases residence time ofsaid nucleic acid in an animal or a human.
 11. The nucleic acidaccording to claim 10, wherein the high molecular weight moiety is a HESor a PEG.
 12. A pharmaceutical composition comprising the nucleic acidaccording to claim 1 and a pharmaceutically acceptable excipient,pharmaceutically acceptable carrier or pharmaceutically active agent.13. A complex comprising the nucleic acid according to claim 1 andeotaxin, MCP-1, MCP-2 or MCP-3.
 14. A method of screening for achemokine antagonist or a chemokine agonist comprising the followingsteps: providing a candidate chemokine antagonist or a candidatechemokine agonist, providing a nucleic acid according to claim 1,providing a test system which provides a signal in the presence of achemokine antagonist or a chemokine agonist, and determining whether thecandidate chemokine antagonist is a chemokine antagonist or whether thecandidate chemokine agonist is a chemokine agonist, wherein thechemokine is eotaxin, MCP-1, MCP-2 or MCP-3.
 15. A method of screeningfor a chemokine agonist or a chemokine antagonist comprising thefollowing steps: providing a chemokine immobilised to a phase, providingthe nucleic acid according to claim 1, optionally which is labelled,adding a candidate chemokine agonist or a candidate chemokineantagonist, and determining whether the candidate chemokine agonist is achemokine agonist or whether the candidate chemokine antagonist is achemokine antagonist, wherein the chemokine is eotaxin, MCP-1, MCP-2 orMCP-3.
 16. A kit for detecting eotaxin, MCP-1, MCP-2or MCP-3 comprisingthe nucleic acid according to claim
 1. 17. A method of detecting thenucleic acid according to claim 1; in a sample, comprising the steps of:a) providing a sample containing the nucleic acid according to claim 1;b) providing a capture probe at least partially complementary to a firstpart of the nucleic acid according to claim 1, and a detection probepartially complementary to a second part of the nucleic acid accordingto claim 1, or, alternatively, the capture probe is at least partiallycomplementary to a second part of the nucleic acid according to claim 1and the detection probe is at least partially complementary to the firstpart of the nucleic acid according to claim 1; c) allowing the captureprobe and the detection probe to react either simultaneously or in anyorder sequentially with the nucleic acid according to claim 1 or partthereof to form a complex of the capture probe, detection probe and thenucleic acid of claim 1; d) optionally detecting whether the captureprobe is hybridized to the nucleic acid according to claim 1 of saidsample; and e) detecting the complex of step c).